Coronavirus vaccine formulations

ABSTRACT

Disclosed herein are coronavirus Spike (S) proteins and nanoparticles comprising the same, which are suitable for use in vaccines. The nanoparticles present antigens from pathogens surrounded to and associated with a detergent core resulting in enhanced stability and good immunogenicity. Dosages, formulations, and methods for preparing the vaccines and nanoparticles are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/997,001, filed Aug. 19, 2020, now U.S. Pat. No. 10,953,089, whichclaims priority to the following applications: U.S. ProvisionalApplication No. 62/966,271, filed Jan. 27, 2020; U.S. ProvisionalApplication No. 62/976,858, filed Feb. 14, 2020; U.S. ProvisionalApplication No. 62/983,180, filed Feb. 28, 2020; U.S. ProvisionalApplication No. 63/048,945, filed Jul. 7, 2020; U.S. ProvisionalApplication No. 63/051,706, filed Jul. 14, 2020; and U.S. ProvisionalApplication No. 63/054,182, filed Jul. 20, 2020. Each of theaforementioned applications is incorporated by reference herein in itsentirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:NOVV_088_09US_SeqList_ST25.txt, date recorded: Mar. 18, 2021; file size:514 kilobytes).

FIELD

The present disclosure is generally related to non-naturally occurringcoronavirus (CoV) Spike (S) polypeptides and nanoparticles and vaccinescomprising the same, which are useful for stimulating immune responses.The nanoparticles provide antigens, for example, glycoprotein antigens,optionally associated with a detergent core and are typically producedusing recombinant approaches. The nanoparticles have improved stabilityand enhanced epitope presentation. The disclosure also providescompositions containing the nanoparticles, methods for producing them,and methods of stimulating immune responses.

BACKGROUND OF THE INVENTION

Infectious diseases remain a problem throughout the world. Whileprogress has been made on developing vaccines against some pathogens,many remain a threat to human health. The outbreak of sudden acuterespiratory syndrome coronavirus 2 (SARS-CoV-2) (also called Wuhancoronavirus and SARS-CoV-2) has infected more than 2000 people in Chinaand killed at least 17 people. Recently, the SARS-CoV-2coronavirus hasspread to the United States, Thailand, South Korea, Taiwan, and Japan.The SARS-CoV-2coronavirus belongs to the same family of viruses assevere acute respiratory syndrome coronavirus (SARS-CoV) and Middle Eastrespiratory syndrome coronavirus (MERS-CoV), which have killed hundredsof people in the past 17 years. SARS-CoV-2 causes the disease COVID-19.

The development of vaccines that prevent or reduce the severity oflife-threatening infectious diseases like the SARS-CoV-2 coronavirus isdesirable. However, human vaccine development remains challengingbecause of the highly sophisticated evasion mechanisms of pathogens anddifficulties stabilizing vaccines. Optimally, a vaccine must both induceantibodies that block or neutralize infectious agents and remain stablein various environments, including environments that do not enablerefrigeration.

SUMMARY OF THE INVENTION

The present disclosure provides non-naturally occurring CoV Spolypeptides suitable for inducing immune responses against SARS-CoV-2(also called Wuhan CoV and 2019-nCoV)). The disclosure also providesnanoparticles containing the glycoproteins as well as methods ofstimulating immune responses.

The present disclosure also provides CoV S polypeptides suitable forinducing immune responses against multiple coronaviruses, includingSARS-CoV-2, Middle East Respiratory Syndrome (MERS), and Severe AcuteRespiratory Syndrome (SARS).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a schematic of the wild-type amino acid sequence of theSARS-CoV-2 Spike (S) protein (SEQ ID NO: 1). The furin cleavage siteRRAR (SEQ ID NO: 6) is highlighted in bold, and the signal peptide isunderlined.

FIG. 2 shows the primary structure of the wild-type CoV S polypeptide,which has an inactive furin cleavage site, a fusion peptide deletion,and K986P and V987P mutations. The domain positions are numbered withrespect to the amino acid sequence of the wild-type CoV S polypeptidefrom SARS-CoV-2 containing a signal peptide (SEQ ID NO: 1).

FIG. 3 shows the primary structure of the BV2378 CoV S polypeptide,which has an inactive furin cleavage site, a fusion peptide deletion,and K986P and V987P mutations. The domain positions are numbered withrespect to the amino acid sequence of the wild-type CoV S polypeptidefrom SARS-CoV-2 containing a signal peptide (SEQ ID NO: 1).

FIG. 4 shows purification of the CoV S polypeptides BV2364, BV2365,BV2366, BV2367, BV2368, BV2369, BV2373, BV2374, and BV2375. The datareveal that BV2365 (SEQ ID NO: 4) and BV2373 (SEQ ID NO: 87) which hasan inactive furin cleavage site having an amino acid sequence of QQAQ(SEQ ID NO: 7) is expressed as a single chain (S0). In contrast, CoV Spolypeptides containing an intact furin cleavage site (e.g. BV2364,BV2366, and BV2374) are cleaved, as evident by the presence of thecleavage product S2.

FIG. 5 shows that the CoV S polypeptides BV2361, BV2365, BV2369, BV2365,BV2373, and BV2374 bind to human angiotensin-converting enzyme 2precursor (hACE2) by bio-layer interferometry.

FIG. 6 shows that BV2361 from SARS-CoV-2 does not bind the MERS-CoVreceptor, dipeptidyl peptidase IV (DPP4) and the MERS S protein does notbind to human angiotensin-converting enzyme 2 precursor (hACE2) bybio-layer interferometry.

FIG. 7 shows that BV2361 binds to hACE2 by enzyme-linked immunosorbentassay (ELISA).

FIG. 8 shows the primary structure of the BV2373 CoV S polypeptide andmodifications to the furin cleavage site, K986P, and V987P.

FIG. 9 shows purification of the wild type CoV S polypeptide and the CoVS polypeptides BV2365 and BV2373.

FIG. 10 shows a cryo-electron microscopy (cryoEM) structure of theBV2373 CoV S polypeptide overlaid on the cryoEM structure of theSARS-CoV-2 spike protein (EMB ID: 21374).

FIGS. 11A-F show that the CoV S Spike polypeptides BV2365 and BV2373bind to hACE2. Bio-layer interferometry reveals that BV2365 (FIG. 11B)and BV2373 (FIG. 11C) bind to hACE2 with similar dissociation kineticsto the wild-type CoV S polypeptide (FIG. 11A) ELISA shows that thewild-type CoV S polypeptide (FIG. 11D) and BV2365 (FIG. 11E) bind tohACE2 with similar affinity while BV2373 binds to hACE2 at a higheraffinity (FIG. 11F).

FIGS. 12A-B show the effect of stress conditions, such as temperature,two freeze/thaw cycles, oxidation, agitation, and pH extremes on bindingof the CoV S polypeptides BV2373 (FIG. 12A) and BV2365 (FIG. 12B) tohACE2.

FIGS. 13A-B show anti-CoV S polypeptide IgG titers 13 days, 21 days, and28 days after immunization of mice with two doses (FIG. 13A) and onedose of 0.1 μg to 10 μg of BV2373 with or without Fraction A andFraction C iscom matrix (e.g., MATRIX-M™) (FIG. 13B).

FIG. 14 shows the induction of antibodies that block interaction ofhACE2 in mice immunized with one dose or two doses of 0.1 μg to 10 μg ofBV2373 with or without MATRIX-M™.

FIG. 15 shows virus neutralizing antibodies detected in mice immunizedwith one dose or two doses of 0.1 μg to 10 μg of BV2373 with or withoutMATRIX-M™.

FIG. 16 shows the virus load (SARS-CoV-2) in the lungs of Ad/CMV/hACE2mice immunized with either a single dose of BV2373 or two doses ofBV2373 spaced 14 days apart with or without MATRIX-M^(Tm).

FIGS. 17A-C shows weight loss exhibited by mice after immunization withBV2373.

FIG. 17A shows the effect of immunization on weight loss with a single0.01 μg, 0.1 μg, 1 μg, or 10 μg of BV2373 plus MATRIX-M™. FIG. 17B showsthe effect of immunization on weight loss with two doses of BV2373 (0.01μg, 0.1 μg, 1 μg) plus MATRIX-M™. FIG. 17C shows the effect ofimmunization on weight loss with two doses of BV2373 (10 μg) in thepresence or absence of MATRIX-M™.

FIGS. 18A-B shows the effect of BV2373 on lung histopathology of micefour days (FIG. 18A) or seven days (FIG. 18B) after infection withSARS-CoV-2.

FIG. 19 shows the number of IFN-γ secreting cells after ex vivostimulation in the spleens of mice immunized with BV2373 in the absenceof adjuvant compared to mice immunized with BV2373 in the presence ofMATRIX-M™.

FIGS. 20A-E shows the frequency of cytokine secreting CD4+ T cells inthe spleens of mice immunized with BV2373 in the presence or absence ofMATRIX-M™. FIG. 20A shows the frequency of IFN-γ secreting CD4+ T cells.FIG. 20B shows the frequency of TNF-α secreting CD4+ T cells. FIG. 20Cshows the frequency of IL-2 secreting CD4+ T cells. FIG. 20D shows thefrequency of CD4+ T cells that secrete two cytokines selected fromIFN-γ, TNF-α, and IL-2. FIG. 20E shows the frequency of CD4+ T cellsthat express IFN-γ, TNF-α, and IL-2.

FIGS. 21A-E shows the frequency of cytokine secreting CD8⁺ T cells inthe spleens of mice immunized with BV2373 in the presence or absence ofMATRIX-M™. FIG. 21A shows the frequency of IFN-γ secreting CD8⁺ T cells.FIG. 21B shows the frequency of TNF-α secreting CD8⁺ T cells. FIG. 21Cshows the frequency of IL-2 secreting CD8⁺ T cells. FIG. 20D shows thefrequency of CD8⁺ T cells that secrete two cytokines selected fromIFN-γ, TNF-α, and IL-2. FIG. 21E shows the frequency of CD8⁺ T cellsthat express IFN-γ, TNF-α, and IL-2.

FIG. 22 illustrates the frequency of CD4⁺ or CD8⁺ cells that express one(single), two (double), or three (triple) cytokines selected from IFN-γ,TNF-α, and IL-2 in the spleens of mice immunized with BV2373 in thepresence or absence of MATRIX-M™.

FIGS. 23A-C illustrate the effect of immunization with BV2373 in thepresence or absence of MATRIX-M™ on type 2 cytokine secretion from CD4⁺T cells. FIG. 23A shows the frequency of IL-4 secreting cells. FIG. 23Bshows the frequency of IL-5 CD4⁺ secreting cells. FIG. 23C shows theratio of IFN-γ secreting to IL-4 secreting CD4⁺ T cells.

FIGS. 24A-B illustrate the effect of mouse immunization with BV2373 inthe presence or absence of MATRIX-M™ on germinal center formation byassessing the presence of CD4⁺ T follicular helper cells (TFH). FIG. 24Ashows the frequency of CD4⁺ T follicular helper cells in spleens, andFIG. 24B shows the phenotype (e.g. CD4⁺CXCR5⁺PD-1⁺) of the CD4⁺ Tfollicular helper cells.

FIGS. 25A-B illustrate the effect of mouse immunization with BV2373 inthe presence or absence of MATRIX-M™ on germinal center formation byassessing the presence of germinal center (GC) B cells. FIG. 25A showsthe frequency of GC B cells in spleens, and FIG. 25B reveals thephenotype (e.g. CD19⁺GL7⁺CD-95⁺) of the CD4⁺ T follicular helper cells.

FIGS. 26A-C show the effect of immunization with BV2373 in the presenceor absence of MATRIX-M™ on antibody response in olive baboons. FIG. 26Ashows the anti-SARS-CoV-2 S polypeptide IgG titer in baboons afterimmunization with BV2373. FIG. 26B shows the presence of hACE2 receptorblocking antibodies in baboons following a single immunization with 5 μgor 25 μg of BV2373 in the presence of MATRIX-M™. FIG. 26C shows thetiter of virus neutralizing antibodies following a single immunizationwith BV2373 and MATRIX-M™.

FIG. 27 shows the significant correlation between anti-SARS-CoV-2 Spolypeptide IgG and neutralizing antibody titers in olive baboons afterimmunization with BV2373.

FIG. 28 shows the frequency of IFN-γ secreting cells in peripheral bloodmononuclear cells (PBMC) of olive baboons immunized with BV2373 in thepresence or absence of MATRIX-M™.

FIGS. 29A-E shows the frequency of cytokine secreting CD4+ T cells inthe PBMC of olive baboons immunized with BV2373 in the presence orabsence of MATRIX-M™. FIG. 29A shows the frequency of IFN-γ secretingCD4+ T cells. FIG. 29B shows the frequency of IL-2 secreting CD4+ Tcells. FIG. 29C shows the frequency of TNF-α secreting CD4+ T cells.FIG. 29D shows the frequency of CD4+ T cells that secrete two cytokinesselected from IFN-γ, TNF-α, and IL-2. FIG. 29E shows the frequency ofCD4+ T cells that express IFN-γ, TNF-α, and IL-2.

FIG. 30 shows a schematic of the coronavirus Spike (S) protein (SEQ IDNO: 109) (BV2384). The furin cleavage site GSAS (SEQ ID NO: 97) isunderlined once, and the K986P and V987P mutations are underlined twice.

FIG. 31 shows a schematic of the coronavirus Spike (S) protein (SEQ IDNO: 86) (BV2373). The furin cleavage site QQAQ (SEQ ID NO: 7) isunderlined once, and the K986P and V987P mutations are underlined twice.

FIG. 32 shows purification of the CoV S polypeptides BV2373 (SEQ ID NO:87) and BV2384 (SEQ ID NO: 109).

FIG. 33 shows a scanning densitometry plot of BV2384 (SEQ ID NO: 109)purity after purification.

FIG. 34 shows a scanning densitometry plot of BV2373 (SEQ ID NO: 87)purity after purification

FIGS. 35A-B illustrates induction of anti-S antibodies (FIG. 35A) andneutralizing antibodies (FIG. 35B) in response to administration ofBV2373 and MATRIX-M™. Cynomolgus macaques were administered one or twodoses (Day 0 and Day 21) of 2.5 μg, 5 μg, or 25 μg of BV2373 with 25 μgor 50 μg MATRIX-M™ adjuvant. Controls received neither BV2373 orMATRIX-M™. Antibodies were measured at Days 21 and 33.

FIGS. 36A-B illustrates a decrease of SARS-CoV-2 viral replication byvaccine formulations disclosed herein as assessed in broncheoalveollavage (BAL) in Cynomolgus macaques. Cynomolgus macaques wereadministered BV2373 and MATRIX-M™ as shown. Subjects were immunized Day0 and in the groups with two doses Day 0 and Day 21. Subject animalswere challenged Day 37 with 1×10⁴ pfu SARS-CoV-2 virus. Viral RNA (FIG.36A, corresponding to total RNA present) and viral sub-genomic RNA (FIG.36B, corresponding to replicating virus) levels were assessed inbronchiolar lavage (BAL) at 2 days and 4 days post-challenge withinfectious virus (d2pi and d4pi). Most subjects showed no viral RNA. AtDay 2 small amounts of RNA were measured in some subjects. By Day 4, noRNA was measured except for two subjects at the lowest dose of 2.5 μg.Sub-genomic RNA was not detected at either 2 Days or 4 days except for 1subject, again at the lowest dose.

FIGS. 37A-B illustrates a decrease of SARS-CoV-2 viral replication byvaccine formulations disclosed herein as assessed in nasal swab inCynomolgus macaques. Cynomolgus macaques were administered BV2373 withMATRIX-M™ as shown. Subjects were immunized Day 0 and in the groups withtwo doses Day 0 and Day 21. Subject animals were challenged Day 37 with1×10⁴ SARS-CoV-2 virus. Viral RNA (FIG. 37A) and viral sub-genomic (sg)RNA (FIG. 37B) were assessed by nasal swab at 2 days and 4 dayspost-infection (d2pi and d4pi). Most subjects showed no viral RNA. AtDay 2 and Day 4 small amounts of RNA were measured in some subjects.Sub-genomic RNA was not detected at either 2 Days or 4 days. Subjectswere immunized Day 0 and in the groups with two doses Day 0 and Day 21.These data show that the vaccine decreases nose total virus RNA by100-1000 fold and sgRNA to undetectable levels, and confirm that immuneresponse to the vaccine will block viral replication and prevent viralspread.

FIGS. 38A-B show anti-CoV S polypeptide IgG titers 21 days and 35 daysafter immunization of Cynomolgus macaques with one dose (FIG. 38A) ortwo doses of BV2373 and 25 μg or 50 μg of MATRIX-M™ (FIG. 38B).

FIGS. 38C-38D shows the hACE2 inhibition titer of Cynomolgus macaques 21days and 35 days after immunization of Cynomolgus macaques with one dose(FIG. 38C) or two doses of BV2373 (5 μg) and MATRIX-M™ (25 μg or 50 μg)(FIG. 38D).

FIG. 38E shows the significant correlation between anti-CoV Spolypeptide IgG titer and hACE2 inhibition titer in Cynomolgus macaquesafter administration of BV2373 and MATRIX-M™. Data is shown for Groups2-6 of Table 4.

FIG. 39 shows the anti-CoV S polypeptide titers and hACE2 inhibitiontiter of Cynomolgus macaques 35 days after immunization with two dosesof BV2373 and MATRIX-M™ or after immunization with convalescent humanserum (Groups 2, 4, and 6) of Table 4. These data show that the anti-CoVS polypeptide and hACE2 inhibition titers of Cynomologus macaquesimmunized with BV2373 and MATRIX-M™ is superior to Cynomolgus macaquesimmunized with convalescent serum.

FIGS. 40A-B shows the SARS-CoV-2 neutralizing titers of Cynomolgusmacaques immunized with BV2373 and MATRIX-M™ as determined by cytopathiceffect (CPE) (FIG. 40A) and plaque reduction neutralization test (PRNT)(FIG. 40B).

FIG. 41 shows administration timings of a clinical trial that evaluatedthe safety and efficacy of a vaccine comprising BV2373 and optionallyMATRIX-M™. AESI denotes an adverse event of special interest. MAEEdenotes a medically attended adverse event, and SAE denotes a seriousadverse event.

FIGS. 42A-B show the local (FIG. 42A) and systemic adverse events (FIG.42B) experienced by patients in a clinical trial which evaluated avaccine comprising BV2373 and MATRIX-M™. Groups A-E are identified inTable 5. The data shows that the vaccine was well tolerated and safe.

FIGS. 43A-B show the anti-CoV S polypeptide IgG (FIG. 43A) andneutralization titers (FIG. 43B) 21 days and 35 days after immunizationof participants in a clinical trial which evaluated a vaccine comprisingBV2373 and MATRIX-M™. Horizontal bars represent interquartile range(IRQ) and median area under the curve, respectively. Whisker endpointsare equal to the maximum and minimum values below or above themedian±1.5 times the IQR. The convalescent serum panel includesspecimens from PCR-confirmed COVID-19 participants from Baylor Collegeof Medicine (29 specimens for ELISA and 32 specimens formicroneutralization (MN IC_(>99)). Severity of COVID-19 is denoted as ared mark for hospitalized patients (including intensive care setting), ablue mark for outpatient-treated patients (sample collected in emergencydepartment), and a green mark for asymptomatic (exposed) patients(sample collected from contact/exposure assessment).

FIGS. 44A-C shows the correlation between anti-CoV S polypeptide IgG andneutralizing antibody titers in patients administered convalescent sera(FIG. 44A), two 25 μg doses of BV2373 (FIG. 44B), and two doses (5 μgand 25 μg) of BV2373 with MATRIX-M™ (FIG. 44C). A strong correlation wasobserved between neutralizing antibody titers and anti-CoV-S IgG titersin patients treated with convalescent sera or with adjuvanted BV2373,but not in patients treated with BV2373 in the absence of adjuvant.

FIGS. 45A-D show the frequencies of antigen-specific CD4⁺ T cellsproducing T helper 1 (Th1) cytokines interferon-gamma (IFN-γ), tumornecrosis factor-alpha (TNF-α), and interleukin (IL)-2 and T helper 2(Th2) cytokines IL-5 and IL-13 indicated cytokines from participants inGroups A (placebo, FIG. 45A), B (25 μg BV2373, FIG. 45B), C (5 μg BV2373and 50 MATRIX-M™, FIG. 45C), and D (25 μg BV2373 and 50 μg MATRIX-M™,FIG. 45D) following stimulation with BV2373. “Any 2” in Th1 cytokinepanel means CD4⁺ T cells that can produce two types of Th1 cytokines atthe same time. “All 3” indicates CD4⁺ T cells that produce IFN-γ, TNF-α,and IL-2 simultaneously. “Both” in Th2 panel means CD4⁺ T cells that canproduce Th2 cytokines IL-5 and IL-13 at the same time.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, and in the appended claims, the singular forms “a”,“an”, and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a protein” canrefer to one protein or to mixtures of such protein, and reference to“the method” includes reference to equivalent steps and/or methods knownto those skilled in the art, and so forth.

As used herein, the term “adjuvant” refers to a compound that, when usedin combination with an immunogen, augments or otherwise alters ormodifies the immune response induced against the immunogen. Modificationof the immune response may include intensification or broadening thespecificity of either or both antibody and cellular immune responses.

As used herein, the term “about” or “approximately” when preceding anumerical value indicates the value plus or minus a range of 10%. Forexample, “about 100” encompasses 90 and 110.

As used herein, the terms “immunogen,” “antigen,” and “epitope” refer tosubstances such as proteins, including glycoproteins, and peptides thatare capable of eliciting an immune response.

As used herein, an “immunogenic composition” is a composition thatcomprises an antigen where administration of the composition to asubject results in the development in the subject of a humoral and/or acellular immune response to the antigen.

As used herein, a “subunit” composition, for example a vaccine, thatincludes one or more selected antigens but not all antigens from apathogen. Such a composition is substantially free of intact virus orthe lysate of such cells or particles and is typically prepared from atleast partially purified, often substantially purified immunogenicpolypeptides from the pathogen. The antigens in the subunit compositiondisclosed herein are typically prepared recombinantly, often using abaculovirus system.

As used herein, “substantially” refers to isolation of a substance (e.g.a compound, polynucleotide, or polypeptide) such that the substanceforms the majority percent of the sample in which it is contained. Forexample, in a sample, a substantially purified component comprises 85%,preferably 85%-90%, more preferably at least 95%-99.5%, and mostpreferably at least 99% of the sample. If a component is substantiallyreplaced the amount remaining in a sample is less than or equal to about0.5% to about 10%, preferably less than about 0.5% to about 1.0%.

The terms “treat,” “treatment,” and “treating,” as used herein, refer toan approach for obtaining beneficial or desired results, for example,clinical results. For the purposes of this disclosure, beneficial ordesired results may include inhibiting or suppressing the initiation orprogression of an infection or a disease; ameliorating, or reducing thedevelopment of, symptoms of an infection or disease; or a combinationthereof.

“Prevention,” as used herein, is used interchangeably with “prophylaxis”and can mean complete prevention of an infection or disease, orprevention of the development of symptoms of that infection or disease;a delay in the onset of an infection or disease or its symptoms; or adecrease in the severity of a subsequently developed infection ordisease or its symptoms.

As used herein an “effective dose” or “effective amount” refers to anamount of an immunogen sufficient to induce an immune response thatreduces at least one symptom of pathogen infection. An effective dose oreffective amount may be determined e.g., by measuring amounts ofneutralizing secretory and/or serum antibodies, e.g., by plaqueneutralization, complement fixation, enzyme-linked immunosorbent(ELISA), or microneutralization assay.

As used herein, the term “vaccine” refers to an immunogenic composition,such as an immunogen derived from a pathogen, which is used to induce animmune response against the pathogen that provides protective immunity(e.g., immunity that protects a subject against infection with thepathogen and/or reduces the severity of the disease or condition causedby infection with the pathogen). The protective immune response mayinclude formation of antibodies and/or a cell-mediated response.Depending on context, the term “vaccine” may also refer to a suspensionor solution of an immunogen that is administered to a subject to produceprotective immunity.

As used herein, the term “subject” includes humans and other animals.Typically, the subject is a human. For example, the subject may be anadult, a teenager, a child (2 years to 14 years of age), an infant(birth to 2 year), or a neonate (up to 2 months). In particular aspects,the subject is up to 4 months old, or up to 6 months old. In someaspects, the adults are seniors about 65 years or older, or about 60years or older. In some aspects, the subject is a pregnant woman or awoman intending to become pregnant. In other aspects, subject is not ahuman; for example a non-human primate; for example, a baboon, achimpanzee, a gorilla, or a macaque. In certain aspects, the subject maybe a pet, such as a dog or cat.

As used herein, the term “pharmaceutically acceptable” means beingapproved by a regulatory agency of a U.S. Federal or a state governmentor listed in the U.S. Pharmacopeia, European Pharmacopeia or othergenerally recognized pharmacopeia for use in mammals, and moreparticularly in humans. These compositions can be useful as a vaccineand/or antigenic compositions for inducing a protective immune responsein a vertebrate.

As used herein, the term “about” means plus or minus 10% of theindicated numerical value.

As used herein, the term “NVX-CoV2373” refers to a vaccine compositioncomprising the BV2373 Spike glycoprotein (SEQ ID NO: 87) and Fraction Aand Fraction C iscom matrix (e.g., MATRIX-M™).

Vaccine Compositions Containing Coronavirus (CoV) Spike (S) Proteins

The disclosure provides non-naturally occurring coronavirus (CoV) Spike(S) polypeptides, nanoparticles containing CoV S polypeptides, andimmunogenic compositions and vaccine compositions containing eithernon-naturally occurring CoV S polypeptides or nanoparticles containingCoV S polypeptides. In embodiments, provided herein are methods of usingCoV S polypeptides, nanoparticles, immunogenic compositions, and vaccinecompositions to stimulate an immune response.

Also provided herein are methods of manufacturing the nanoparticles andvaccine compositions. Advantageously, the methods provide nanoparticlesthat are substantially free from contamination by other proteins, suchas proteins associated with recombinant expression of proteins in insectcells. In embodiments, expression occurs in baculovirus/Sf9 systems.

CoV S Polypeptide Antigens

The vaccine compositions of the disclosure contain non-naturallyoccurring CoV S polypeptides. CoV S polypeptides may be derived fromcoronaviruses, including but not limited to SARS-CoV-2, for example fromSARS-CoV-2, from MERS CoV, and from SARS CoV. In contrast to theSARS-CoV S protein, the SARS-CoV-2 S protein has a four amino acidinsertion in the S1/S2 cleavage site resulting in a polybasic RRARfurin-like cleavage motif. The SARS-CoV-2 S protein is synthesized as aninactive precursor (S0) that is proteolytically cleaved at the furincleavage site into 51 and S2 subunits which remain non-covalently linkedto form prefusion trimers. The S2 domain of the SARS-CoV-2 S proteincomprises a fusion peptide (FP), two heptad repeats (HR1 and HR2), atransmembrane (TM) domain, and a cytoplasmic tail. The 51 domain of theSARS-CoV-2 S protein folds into four distinct domains: the N-terminaldomain (NTD) and the C-terminal domain, which contains the receptorbinding domain (RBD) and two subdomains SD1 and SD2. The prefusionSARS-CoV-2 S protein trimers undergo a structural rearrangement from aprefusion to a postfusion conformation upon S-protein receptor bindingand cleavage.

In embodiments, the CoV S polypeptides are glycoproteins, due topost-translational glycosylation. The glycoproteins comprise one or moreof an NTD, an RBD, an SD1/SD2 portion a UH domain, an intact or modifiedfusion protein region, an HR1 domain an HR2 domain, and a TM domain. Inembodiments, the amino acids for each domain are given in FIG. 2 andFIG. 3 (shown corresponding to SEQ ID NO: 1). In embodiments, eachdomain may have at least 95%, at least 97% or at least 99% identity tothe sequences for each domain as in SEQ ID NO: 1. Each domain may have adeletion or an insertion of about 10, about 20, or about 30 amino acidscompared to those shown in SEQ ID NO: 1. Note that FIGS. 2 and 3illustrate the 13-amino acid N-terminal signal peptide that is absentfrom the mature peptide. The CoV S polypeptides may be used to stimulateimmune responses against the native CoV Spike (S) polypeptide.

In embodiments, the native CoV Spike (S) polypeptide (SEQ ID NO: 2) ismodified resulting in non-naturally occurring CoV Spike (S) polypeptides(FIG. 1). In embodiments, the CoV Spike (S) glycoproteins comprise oneor more modifications selected from the group consisting of:

(a) an inactivated mutated furin cleavage site amino acids 669-672;

(b) a deletion of one or more amino acids from amino acids 676-685;

(c) a deletion of one or more amino acids from amino acids 702-711;

(d) a deletion of one or more amino acids of the fusion peptide (aminoacids 806-815);

(e) mutation of amino acid 601;

(f) mutation of amino acid 973;

(g) mutation of amino acid 974;

-   -   (h) a deletion of one or more amino acids from the N-terminal        domain (NTD) (amino acids 1-318); and

(i) a deletion of one or more amino acids from the transmembrane andcytoplasmic domain (TMCT) (amino acids 1201-1260),

wherein the amino acids of the CoV S glycoprotein are numbered withrespect to SEQ ID NO: 2. FIG. 3 shows a CoV S polypeptide called BV2378,which has an inactive furin cleavage site, deleted fusion peptide, aK986P, and a V987 mutation.

In embodiments, the CoV S polypeptides described herein exist in aprefusion conformation. In embodiments, the CoV S polypeptides describedherein comprise a flexible HR2 domain. Unless otherwise mentioned, theflexibility of a domain is determined by transition electron microscopy(TEM) and 2D class averaging. A reduction in electron densitycorresponds to a flexible domain.

In embodiments, the CoV S polypeptides contain a furin site (RRAR),amino acids 669 to 672 of the native CoV Spike (S) polypeptide (SEQ IDNO: 2), that is inactivated by one or more mutations. Inactivation ofthe furin cleavage site prevents furin from cleaving the CoV Spolypeptide. In embodiments, the CoV S polypeptides described hereinwhich contain an inactivated furin cleavage site are expressed as asingle chain.

In embodiments, one or more of the amino acids comprising the nativefurin cleavage site is mutated to any natural amino acid. Inembodiments, the amino acids are L-amino acids. Non-limiting examples ofamino acids include alanine, arginine, glycine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, serine, threonine, histidine,lysine, methionine, proline, valine, isoleucine, leucine, tyrosine,tryptophan, and phenylalanine.

In embodiments, one or more of the amino acids comprising the nativefurin cleavage site is mutated to glutamine. In embodiments, 1, 2, 3, or4 amino acids may be mutated to glutamine. In embodiments, one of thearginines comprising the native furin cleavage site is mutated toglutamine. In embodiments, two of the arginines comprising the nativefurin cleavage site are mutated to glutamine. In embodiments, three ofthe arginines comprising the native furin cleavage site are mutated toglutamine.

In embodiments, one or more of the amino acids comprising the nativefurin cleavage site, is mutated to alanine. In embodiments, 1, 2, 3, or4 amino acids may be mutated to alanine. embodiments, one of thearginines comprising the native furin cleavage site is mutated toalanine. In embodiments, two of the arginines comprising the nativefurin cleavage site are mutated to alanine. In embodiments, three of thearginines comprising the native furin cleavage site are mutated toalanine.

In embodiments, one or more of the amino acids comprising the nativefurin cleavage site is mutated to glycine. In embodiments, 1, 2, 3, or 4amino acids may be mutated to glycine. In embodiments, one of thearginines of the native furin cleavage site is mutated to glycine. Inembodiments, two of the arginines comprising the native furin cleavagesite are mutated to glycine. In embodiments, three of the argininescomprising the native furin cleavage site are mutated to glycine.

In embodiments, one or more of the amino acids comprising the nativefurin cleavage site, is mutated to asparagine. For example 1, 2, 3, or 4amino acids may be mutated to asparagine. In embodiments, one of thearginines comprising the native furin cleavage site is mutated toasparagine. In embodiments, two of the arginines comprising the nativefurin cleavage site are mutated to asparagine. In embodiments, three ofthe arginines comprising the native furin cleavage site are mutated toasparagine.

Non-limiting examples of the amino acid sequences of the inactivatedfurin sites contained within the CoV S polypeptides are found in Table1.

TABLE 1 Inactivated Furin Cleavage Sites Amino Acid Sequence ofActive or Inactive Furin Cleavage Site Furin Cleavage SiteRRAR (SEQ ID NO: 6) Active QQAQ (SEQ ID NO: 7) InactiveQRAR (SEQ ID NO: 8) Inactive RQAR (SEQ ID NO: 9) InactiveRRAQ (SEQ ID NO: 10) Inactive QQAR (SEQ ID NO: 11) InactiveRQAQ (SEQ ID NO: 12) Inactive QRAQ (SEQ ID NO: 13) InactiveNNAN (SEQ ID NO: 14) Inactive NRAR (SEQ ID NO: 15) InactiveRNAR (SEQ ID NO: 16) Inactive RRAN (SEQ ID NO: 17) InactiveNNAR (SEQ ID NO: 18) Inactive RNAN (SEQ ID NO: 19) InactiveNRAN (SEQ ID NO: 20) Inactive AAAA (SEQ ID NO: 21) InactiveARAR (SEQ ID NO: 22) Inactive RAAR (SEQ ID NO: 23) InactiveRRAA (SEQ ID NO: 24) Inactive AAAR (SEQ ID NO: 25) InactiveRAAA (SEQ ID NO: 26) Inactive ARAA (SEQ ID NO: 27) InactiveGGAG (SEQ ID NO: 28) Inactive GRAR (SEQ ID NO: 29) InactiveRGAR (SEQ ID NO: 30) Inactive RRAG (SEQ ID NO: 31) InactiveGGAR (SEQ ID NO: 32) Inactive RGAG (SEQ ID NO: 33) InactiveGRAG (SEQ ID NO: 34) Inactive GSAS (SEQ ID NO: 97) InactiveGSGA (SEQ ID NO: 113) Inactive

In embodiments, in lieu of an active furin cleavage site (SEQ ID NO: 6)the CoV S polypeptides described herein contain an inactivated furincleavage site. In embodiments, the amino acid sequence of theinactivated furin cleavage site is represented by any one of SEQ ID NO:7-34 or SEQ ID NO: 97. In embodiments, the amino acid sequence of theinactivated furin cleavage site is QQAQ (SEQ ID NO: 7). In embodiments,the amino acid sequence of the inactivated furin cleavage site is GSAS(SEQ ID NO: 97). In embodiments, the amino acid sequence of theinactivated furin cleavage site is GSGA (SEQ ID NO: 113).

In embodiments, the CoV S polypeptides contain a deletion, correspondingto one or more deletions within amino acids 676-685 of the native CoVSpike (S) polypeptide (SEQ ID NO: 2). In embodiments, 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acids of amino acids 676-685 of the native CoVSpike (S) polypeptide (SEQ ID NO:2) are deleted. In embodiments, thedeletions of amino acids within amino acids 676-685 are consecutive e.g.amino acids 676 and 677 are deleted or amino acids 680 and 681 aredeleted. In embodiments, the deletions of amino acids within amino acids676-685 are non-consecutive e.g. amino acids 676 and 680 are deleted oramino acids 677 and 682 are deleted. In embodiments, CoV S polypeptidescontaining a deletion, corresponding to one or more deletions withinamino acids 676-685, have an amino acid sequence selected from the groupconsisting of SEQ ID NO: 62 and SEQ ID NO: 63.

In embodiments, the CoV S polypeptides contain a deletion, correspondingto one or more deletions within amino acids 702-711 of the native CoVSpike (S) polypeptide (SEQ ID NO: 2). In embodiments, 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acids of amino acids 702-711 of the nativeSARS-CoV-2 Spike (S) polypeptide (SEQ ID NO:2) are deleted. Inembodiments, the one or more deletions of amino acids within amino acids702-711 are consecutive e.g. amino acids 702 and 703 are deleted oramino acids 708 and 709 are deleted. In embodiments, the deletions ofamino acids within amino acids 702-711 are non-consecutive e.g. aminoacids 702 and 704 are deleted or amino acids 707 and 710 are deleted. Inembodiments, the CoV S polypeptides containing a deletion, correspondingto one or more deletions within amino acids 702-711, have an amino acidsequence selected from the group consisting of SEQ ID NO: 64 and SEQ IDNO: 65.

In embodiments, the CoV S polypeptides contain a deletion of the fusionpeptide (SEQ ID NO: 104), which corresponds to amino acids 806-815 ofSEQ ID NO: 2. In embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids of the fusion peptide of the CoV Spike (S) polypeptide (SEQ IDNO:2) are deleted. In embodiments, the deletions of amino acids withinthe fusion peptide are consecutive e.g. amino acids 806 and 807 aredeleted or amino acids 809 and 810 are deleted. In embodiments, thedeletions of amino acids within the fusion peptide are non-consecutivee.g. amino acids 806 and 808 are deleted or amino acids 810 and 813 aredeleted. In embodiments, the CoV S polypeptides containing a deletion,corresponding to one or more amino acids of the fusion peptide, have anamino acid sequence selected from SEQ ID NOS: 66, 77, and 105-108.

In embodiments, the CoV S polypeptides contain a deletion of one or moreamino acids from the N-terminal domain (NTD) (corresponding to aminoacids 1-318 of SEQ ID NO: 2. The amino acid sequence of the NTD isrepresented by SEQ ID NO: 45. In embodiments, the CoV S polypeptidescontain a deletion of amino acids 1-318 of the N-terminal domain (NTD)of SEQ ID NO: 2. In embodiments, deletion of the NTD enhances proteinexpression of the CoV Spike (S) polypeptide. In embodiments, the CoV Spolypeptides which have an NTD deletion have amino acid sequencesrepresented by SEQ ID NOS: 46, 48, 49, 51, 52, and 54. In embodiments,the CoV S polypeptides which have an NTD deletion are encoded by anisolated nucleic acid sequence selected from the group consisting of SEQID NO: 47, SEQ ID NO: 50, and SEQ ID NO: 53.

In embodiments, the CoV Spike (S) polypeptides contain a deletion of oneor more amino acids from the transmembrane and cytoplasmic domain (TMCT)(corresponding to amino acids 1201-1260). The amino acid sequence of theTMCT is represented by SEQ ID NO: 39. In embodiments, the CoV Spolypeptides which have a deletion of one or more residues of the TMCThave enhanced protein expression. In embodiments, the CoV Spike (S)polypeptides which have one or more deletions from the TMCT have anamino acid sequence selected from the group consisting of SEQ ID NO: 40,41, 42, 52, 54, 59, 61, 88, and 89. In embodiments, the CoV Spolypeptides which have one or more deletions from the TMCT are encodedby an isolated nucleic acid sequence selected from the group consistingof SEQ ID NO: 39, 43, 53, and 60.

In embodiments, the CoV S polypeptides contain a mutation at Asp-601 ofthe native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments,Asp-601 is mutated to any natural amino acid. In embodiments, Asp-601 ismutated to glycine.

In embodiments, the CoV S polypeptides contain mutations that stabilizethe prefusion conformation of the CoV S polypeptide. In embodiments, theCoV S polypeptides contain proline substitutions which stabilize theprefusion conformation. This strategy has been utilized for to develop aprefusion stabilized MERS-CoV S protein as described in the followingdocuments which are each incorporated by reference herein in theirentirety: Proc Natl Acad Sci USA. 2017 August 29; 114(35):E7348-E7357;Sci Rep. 2018 October 24; 8(1):15701; U.S. Publication No. 2020/0061185;and PCT Application No. PCT/US2017/058370.

In embodiments, the CoV S polypeptides contain a mutation at Lys-973 ofthe native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments,Lys-973 is mutated to any natural amino acid. In embodiments, Lys-973 ismutated to proline. In embodiments, the CoV S polypeptides containing amutation at amino acid 973 are selected from the group consisting of SEQID NO: 84-89, 105-106, and 109-110.

In embodiments, the CoV S polypeptides contain a mutation at Val-974 ofthe native CoV Spike (S) polypeptide (SEQ ID NO: 2). In embodiments,Val-974 is mutated to any natural amino acid. In embodiments, Val-974 ismutated to proline. In embodiments, the CoV S polypeptides containing amutation at amino acid 974 are selected from the group consisting of SEQID NO: 84-89, 105-106, and 109-110.

In embodiments, the CoV S polypeptides contain a mutation at Lys-973 andVal-974 of the native CoV Spike (S) polypeptide (SEQ ID NO: 2). Inembodiments, Lys-973 and Val-974 are mutated to any natural amino acid.In embodiments, Lys-973 and Val-974 are mutated to proline. Inembodiments, the CoV S polypeptides containing a mutation at amino acids973 and 974 are selected from SEQ ID NOS: 84-89, 105-106, and 109-110.

In embodiments, the CoV S polypeptides contain a mutation at Lys-973 andVal-974 and an inactivated furin cleavage site. In embodiments, the CoVS polypeptides contain mutations of Lys-973 and Val-974 to proline andan inactivated furin cleavage site, having the amino acid sequence ofQQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). An exemplary CoV Spolypeptide containing a mutation at Lys-973 and Val-974 and aninactivated furin cleavage site is depicted in FIG. 8. In embodiments,the CoV S polypeptides containing mutations of Lys-973 and Val-974 toproline and an inactivated furin cleavage site have an amino acidsequences of SEQ ID NOS: 86 or 87 and a nucleic acid sequence of SEQ IDNO: 96.

In embodiments, the CoV S polypeptides contain a mutation at Lys-973 andVal-974, an inactivated furin cleavage site, and a deletion of one ormore amino acids of the fusion peptide. In embodiments, the CoV Spolypeptides contain mutations of Lys-973 and Val-974 to proline, aninactivated furin cleavage site having the amino acid sequence of QQAQ(SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), and deletion of one or moreamino acids of the fusion peptide. In embodiments, the CoV Spolypeptides containing mutations of Lys-973 and Val-974 to proline, aninactivated furin cleavage site, and deletion of one or more amino acidsof the fusion peptide has an amino acid sequence of SEQ ID NO: 105 or106.

In embodiments, the CoV Spike (S) polypeptides comprise a polypeptidelinker. In embodiments, the polypeptide linker contains glycine andserine. In embodiments, the linker has about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, or about 100% glycine.

In embodiments, the polypeptide linker has a repeat of (SGGG)_(n) (SEQID NO: 91), wherein n is an integer from 1 to 50 (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50). In embodiments, the polypeptide linkerhas an amino acid sequence corresponding to SEQ ID NO: 90.

In embodiments, the polypeptide linker has a repeat of (GGGGS)_(n) (SEQID NO: 93), wherein n is an integer from 1 to 50 (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50).

In embodiments, the polypeptide linker has a repeat of (GGGS)_(n) (SEQID NO: 92), wherein n is an integer from 1 to 50 (e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50).

In some aspects, the polypeptide linker is a poly-(Gly)_(n) linker,wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, or 20.In other embodiments, the linker is selected from the group consistingof: dipeptides, tripeptides, and quadripeptides. In embodiments, thelinker is a dipeptide selected from the group consisting ofalanine-serine (AS), leucine-glutamic acid (LE), and serine-arginine(SR).

In embodiments, the polypeptide linker comprises between 1 to 100contiguous amino acids of a naturally occurring CoV S polypeptide or ofa CoV S polypeptide disclosed herein. In embodiments, the polypeptidelinker has an amino acid sequence corresponding to SEQ ID NO: 94.

In embodiments, the CoV Spike (S) polypeptides comprise a foldon. Inembodiments, the TMCT is replaced with a foldon. In embodiments, afoldon causes trimerization of the CoV Spike (S) polypeptide. Inembodiments, the foldon is an amino acid sequence known in the art. Inembodiments, the foldon has an amino acid sequence of SEQ ID NO: 68. Inembodiments, the foldon is a T4 fibritin trimerization motif. Inembodiments, the T4 fibritin trimerization domain has an amino acidsequence of SEQ ID NO: 103. In embodiments, the foldon is separated inamino acid sequence from the CoV Spike (S) polypeptide by a polypeptidelinker. Non-limiting examples of polypeptide linkers are foundthroughout this disclosure.

In embodiments, the disclosure provides CoV S polypeptides comprising afragment of a coronavirus S protein and nanoparticles and vaccinescomprising the same. In embodiments, the fragment of the coronavirus Sprotein is between 10 and 1500 amino acids in length (e.g. about 10,about 20, about 30, about 40, about 50, about 60, about 70, about 80,about 90, about 100, about 150, about 200, about 250, about 300, about350, about 400, about 450, about 500, about 550, about 600, about 650,about 700, about 750, about 800, about 850, about 900, about 950, about1000, about 1050, about 1100, about 1150, about 1200, about 1250, about1300, about 1350, about 1400, about 1450, or about 1500 amino acids inlength). In embodiments, the fragment of the coronavirus S protein isselected from the group consisting of the receptor binding domain (RBD),subdomain 1, subdomain 2, upper helix, fusion peptide, connectingregion, heptad repeat 1, central helix, heptad repeat 2, NTD, and TMCT.

In embodiments, the CoV S polypeptide comprises an RBD and asubdomain 1. In embodiments, the CoV S polypeptide comprising an RBD anda subdomain 1 is amino acids 319 to 591 of SEQ ID NO: 1.

In embodiments, the CoV S polypeptide contains a fragment of acoronavirus S protein, wherein the fragment of the coronavirus S proteinis the RBD. Non-limiting examples of RBDs include the RBD of SARS-CoV-2(amino acid sequence=SEQ ID NO: 69), the RBD of SARS (amino acidsequence=SEQ ID NO: 70), and the RBD of MERS, (amino acid sequence=SEQID NO: 71).

In embodiments, the CoV S polypeptide contains two or more RBDs, whichare connected by a polypeptide linker. In embodiments, the polypeptidelinker has an amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 94.

In embodiments, the CoV S polypeptide contains 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 RBDs.

In some embodiments, the CoV S polypeptide contains two or moreSARS-CoV-2 RBDs, which are connected by a polypeptide linker. Inembodiments, the antigen containing two or more SARS-CoV-2 RBDs has anamino acid sequence corresponding to one of SEQ ID NOS: 72-75.

In embodiments, the CoV S polypeptide contains a SARS-CoV-2 RBD and aSARS RBD. In embodiments, the CoV S polypeptide comprises a SARS-CoV-2RBD and a SARS RBD, wherein each RBD is separated by a polypeptidelinker. In embodiments, the CoV S polypeptide comprising a SARS-CoV-2RBD and a SARS RBD has an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 76-79.

In embodiments, the CoV S polypeptide contains a SARS-CoV-2 RBD and aMERS RBD. In embodiments, the CoV S polypeptide comprises a SARS-CoV-2RBD and a MERS RBD, wherein each RBD is separated by a polypeptidelinker.

In embodiments, the CoV S polypeptide comprises a SARS RBD and a MERSRBD. In embodiments, the CoV S polypeptide comprises a SARS RBD and aMERS RBD, wherein each RBD is separated by a polypeptide linker.

In embodiments, the CoV S polypeptide contains a SARS-CoV-2 RBD, a SARSRBD, and a MERS RBD. In embodiments, the CoV S polypeptide contains aSARS-CoV-2 RBD, a SARS RBD, and a MERS RBD, wherein each RBD isseparated by a polypeptide linker. In embodiments, the CoV S polypeptidecomprising a SARS-CoV-2 RBD, a SARS RBD, and a MERS RBD has an aminoacid sequence selected from the group consisting of SEQ ID NOS: 80-83.

In embodiments, the CoV S polypeptides described herein are expressedwith an N-terminal signal peptide. In embodiments, the N-terminal signalpeptide consists of an amino acid sequence of SEQ ID NO: 5(MFVFLVLLPLVSS). In embodiments, the signal peptide may be replaced withany signal peptide that enables expression of the CoV S protein. Inembodiments, one or more of the CoV S protein signal peptide amino acidsmay be deleted or mutated. An initiating methionine residue ismaintained to initiate expression. In embodiments, the CoV Spolypeptides are encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 95, SEQ IDNO: 43, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 55, SEQID NO: 57, SEQ ID NO: 96, and SEQ ID NO: 60.

Following expression of the CoV S protein in a host cell, the N-terminalsignal peptide is cleaved to provide the mature CoV protein sequence(SEQ ID NOS: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75,78, 79, 82, 83, 85, 87, 89, 106, and 110). In embodiments, the signalpeptide is cleaved by host cell proteases. In aspects, the full-lengthprotein may be isolated from the host cell and the signal peptidecleaved subsequently.

Following cleavage of the signal peptide from the CoV Spike (S)polypeptide with an amino acid sequence corresponding to SEQ ID NOS: 1,3, 36, 40, 42, 46, 49, 52, 56, 59, 62, 64, 66, 72, 74, 76, 77, 80, 81,84, 86, 87, 105, 107, 88, and 109 during expression and purification, amature polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65,67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89, and 110 is obtained andused to produce a CoV S nanoparticle vaccine or CoV S nanoparticles.

Advantageously, the disclosed CoV S polypeptides may have enhancedprotein expression and stability relative to the native CoV Spike (S)protein.

In embodiments, the CoV S polypeptides described herein contain furthermodifications from the native coronavirus S protein (SEQ ID NO: 2). Inembodiments, the coronavirus S proteins described herein exhibit atleast 80%, or at least 90%, or at least 95%, or at least 97%, or atleast 99% identity to the native coronavirus S protein. A person ofskill in the art would use known techniques to calculate the percentidentity of the recombinant coronavirus S protein to the native protein.For example, percentage identity can be calculated using the toolsCLUSTALW2 or Basic Local Alignment Search Tool (BLAST), which areavailable online. The following default parameters may be used forCLUSTALW2 Pairwise alignment: Protein Weight Matrix=Gonnet; Gap Open=10;Gap Extension=0.1.

In embodiments, the CoV S polypeptides described herein comprise about1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about9, about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, or about 25 substitutions compared to the coronavirus Sprotein (SEQ ID NO: 87).

In embodiments, the coronavirus S polypeptide is extended at theN-terminus, the C-terminus, or both the N-terminus and the C-terminus.In some aspects, the extension is a tag useful for a function, such aspurification or detection. In some aspects the tag contains an epitope.For example, the tag may be a polyglutamate tag, a FLAG-tag, a HA-tag, apolyHis-tag (having about 5-10 histidines) (SEQ ID NO: 101), ahexahistidine tag (SEQ ID NO: 100), an 8X-His-tag (having eighthistidines) (SEQ ID NO: 102), a Myc-tag, aGlutathione-S-transferase-tag, a Green fluorescent protein-tag, Maltosebinding protein-tag, a Thioredoxin-tag, or an Fc-tag. In other aspects,the extension may be an N-terminal signal peptide fused to the proteinto enhance expression. While such signal peptides are often cleavedduring expression in the cell, some nanoparticles may contain theantigen with an intact signal peptide. Thus, when a nanoparticlecomprises an antigen, the antigen may contain an extension and thus maybe a fusion protein when incorporated into nanoparticles. For thepurposes of calculating identity to the sequence, extensions are notincluded. In embodiments, the tag is a protease cleavage site.Non-limiting examples of protease cleavage sites include the HRV3Cprotease cleavage site, chymotrypsin, trypsin, elastase, endopeptidase,caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6,caspase-7, caspase-8, caspase-9, caspase-10, enterokinase, factor Xa,Granzyme B, TEV protease, and thrombin. In embodiments, the proteasecleavage site is an HRV3C protease cleavage site. In embodiments, theprotease cleavage site comprises an amino acid sequence of SEQ ID NO:98.

In embodiments, the CoV S glycoprotein comprises a fusion protein. Inembodiments, the CoV S glycoprotein comprises an N-terminal fusionprotein. In embodiments, the Cov S glycoprotein comprises a C-terminalfusion protein. In embodiments, the fusion protein encompasses a taguseful for protein expression, purification, or detection. Inembodiments, the tag is a polyHis-tag (having about 5-10 histidines), aMyc-tag, a Glutathione-S-transferase-tag, a Green fluorescentprotein-tag, Maltose binding protein-tag, a Thioredoxin-tag, aStrep-tag, a Twin-Strep-tag, or an Fc-tag. In embodiments, the tag is anFc-tag. In embodiments, the Fc-tag is monomeric, dimeric, or trimeric.In embodiments, the tag is a hexahistidine tag, e.g. a polyHis-tag whichcontains six histidines (SEQ ID NO: 100). In embodiments, the tag is aTwin-Strep-tag with an amino acid sequence of SEQ ID NO: 99.

In embodiments, the CoV S polypeptide is a fusion protein comprisinganother coronavirus protein. In embodiments, the other coronavirusprotein is from the same coronavirus. In embodiments, the othercoronavirus protein is from a different coronavirus.

In some aspects, the CoV S protein may be truncated. For example, theN-terminus may be truncated by about 10 amino acids, about 30 aminoacids, about 50 amino acids, about 75 amino acids, about 100 aminoacids, or about 200 amino acids. The C-terminus may be truncated insteadof or in addition to the N-terminus. For example, the C-terminus may betruncated by about 10 amino acids, about 30 amino acids, about 50 aminoacids, about 75 amino acids, about 100 amino acids, or about 200 aminoacids. For purposes of calculating identity to the protein havingtruncations, identity is measured over the remaining portion of theprotein.

Nanoparticles Containing CoV Spike (S) Polypeptides

In embodiments, the mature CoV S polypeptide antigens are used toproduce a vaccine comprising coronavirus S nanoparticles. Inembodiments, nanoparticles of the present disclosure comprise the CoV Spolypeptides described herein. In embodiments, the nanoparticles of thepresent disclosure comprise CoV S polypeptides associated with adetergent core. The presence of the detergent facilitates formation ofthe nanoparticles by forming a core that organizes and presents theantigens. In embodiments, the nanoparticles may contain the CoV Spolypeptides assembled into multi-oligomeric glycoprotein-detergent(e.g.PS80) nanoparticles with the head regions projecting outward andhydrophobic regions and PS80 detergent forming a central core surroundedby the glycoprotein. In embodiments, the CoV S polypeptide inherentlycontains or is adapted to contain a transmembrane domain to promoteassociation of the protein into a detergent core. In embodiments, theCoV S polypeptide contains a head domain. FIG. 10 shows an exemplarystructure of a CoV S polypeptide of the disclosure. Primarily thetransmembrane domains of a CoV S polypeptide trimer associate withdetergent; however, other portions of the polypeptide may also interact.Advantageously, the nanoparticles have improved resistance toenvironmental stresses such that they provide enhanced stability and/orimproved presentation to the immune system due to organization ofmultiple copies of the protein around the detergent.

In embodiments, the detergent core is a non-ionic detergent core. Inembodiments, the CoV S polypeptide is associated with the non-ionicdetergent core. In embodiments, the detergent is selected from the groupconsisting of polysorbate-20 (PS20), polysorbate-40 (PS40),polysorbate-60 (PS60), polysorbate-65 (PS65) and polysorbate-80 (PS80).

In embodiments, the detergent is PS80.

In embodiments, the CoV S polypeptide forms a trimer. In embodiments,the CoV S polypeptide nanoparticles are composed of multiple polypeptidetrimers surrounding a non-ionic detergent core. In embodiments, thenanoparticles contain at least about 1 trimer or more. In embodiments,the nanoparticles contain at least about 5 trimers to about 30 trimersof the Spike protein. In embodiments, each nanoparticle may contain 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15, 20, 25, or 30 trimers,including all values and ranges in between. Compositions disclosedherein may contain nanoparticles having different numbers of trimers.For example, a composition may contain nanoparticles where the number oftrimers ranges from 2-9; in embodiments, the nanoparticles in acomposition may contain from 2-6 trimers. In embodiments, thecompositions contain a heterogeneous population of nanoparticles having2 to 6 trimers per nanoparticle, or 2 to 9 trimers per nanoparticle. Inembodiments, the compositions may contain a substantially homogenouspopulation of nanoparticles. For example, the population may containabout 95% nanoparticles having 5 trimers.

The nanoparticles disclosed herein range in particle size. Inembodiments, the nanoparticles disclosed herein range in particle sizefrom a Z-ave size from about 20 nm to about 60 nm, about 20 nm to about50 nm, about 20 nm to about 45 nm, about 20 nm to about 35 nm, about 20nm to about 30 nm, about 25 nm to about 35 nm, or about 25 nm to about45 nm. Particle size (Z-ave) is measured by dynamic light scattering(DLS) using a Zetasizer NanoZS (Malvern, UK), unless otherwisespecified.

In embodiments, the nanoparticles comprising the CoV S polypeptidesdisclosed herein have a reduced particle size compared to nanoparticlescomprising a wild-type CoV S polypeptide. In embodiments, the CoV Spolypeptides are at least about 40% smaller in particle size, forexample, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, or at least about 85%smaller in particle size.

The nanoparticles comprising CoV S polypeptides disclosed herein aremore homogenous in size, shape, and mass than nanoparticles comprising awild-type CoV S polypeptide. The polydispersity index (PDI), which is ameasure of heterogeneity, is measured by dynamic light scattering usinga Malvern Setasizer unless otherwise specified. In embodiments, theparticles measured herein have a PDI from about 0.2 to about 0.45, forexample, about 0.2, about 0.25, about 0.29, about 0.3, about 0.35, about0.40, or about 0.45. In embodiments, the nanoparticles measured hereinhave a PDI that is at least about 25% smaller than the PDI ofnanoparticles comprising the wild-type CoV S polypeptide, for example,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,or at least about 60%, smaller.

The CoV S polypeptides and nanoparticles comprising the same haveimproved thermal stability as compared to the wild-type CoV Spolypeptide or a nanoparticle thereof. The thermal stability of the CoVS polypeptides is measured using differential scanning calorimetry (DSC)unless otherwise specified. The enthalpy of transition (ΔHcal) is theenergy required to unfold a CoV S polypeptide. In embodiments, the CoV Spolypeptides have an increased ΔHcal as compared to the wild-type CoV Spolypeptide. In embodiments, the ΔHcal of a CoV S polypeptide is about2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about7-fold, about 8-fold, about 9-fold, or about 10-fold greater than theΔHcal of a wild-type CoV S polypeptide.

Several nanoparticle types may be included in vaccine compositionsdisclosed herein. In some aspects, the nanoparticle type is in the formof an anisotropic rod, which may be a dimer or a monomer. In otheraspects, the nanoparticle type is a spherical oligomer. In yet otheraspects, the nanoparticle may be described as an intermediatenanoparticle, having sedimentation properties intermediate between thefirst two types. Formation of nanoparticle types may be regulated bycontrolling detergent and protein concentration during the productionprocess. Nanoparticle type may be determined by measuring sedimentationco-efficient.

Production of Nanoparticles Containing CoV S Polypeptide Antigens

The nanoparticles of the present disclosure are non-naturally occurringproducts, the components of which do not occur together in nature.Generally, the methods disclosed herein use a detergent exchangeapproach wherein a first detergent is used to isolate a protein and thenthat first detergent is exchanged for a second detergent to form thenanoparticles.

The antigens contained in the nanoparticles are typically produced byrecombinant expression in host cells. Standard recombinant techniquesmay be used. In embodiments, the CoV S polypeptides are expressed ininsect host cells using a baculovirus system. In embodiments, thebaculovirus is a cathepsin-L knock-out baculovirus, a chitinaseknock-out baculovirus. Optionally, the baculovirus is a double knock-outfor both cathepsin-L and chitinase. High level expression may beobtained in insect cell expression systems. Non limiting examples ofinsect cells are, Spodoptera frugiperda (Sf) cells, e.g. Sf9, Sf21,Trichoplusiani cells, e.g. High Five cells, and Drosophila S2 cells. Inembodiments, the CoV S polypeptide described herein are produced in anysuitable host cell. In embodiments, the host cell is an insect cell. Inembodiments, the insect cell is an Sf9 cell.

Typical transfection and cell growth methods can be used to culture thecells. Vectors, e.g., vectors comprising polynucleotides that encodefusion proteins, can be transfected into host cells according to methodswell known in the art. For example, introducing nucleic acids intoeukaryotic cells can be achieved by calcium phosphate co-precipitation,electroporation, microinjection, lipofection, and transfection employingpolyamine transfection reagents. In one embodiment, the vector is arecombinant baculovirus.

Methods to grow host cells include, but are not limited to, batch,batch-fed, continuous and perfusion cell culture techniques. Cellculture means the growth and propagation of cells in a bioreactor (afermentation chamber) where cells propagate and express protein (e.g.recombinant proteins) for purification and isolation. Typically, cellculture is performed under sterile, controlled temperature andatmospheric conditions in a bioreactor. A bioreactor is a chamber usedto culture cells in which environmental conditions such as temperature,atmosphere, agitation and/or pH can be monitored. In one embodiment, thebioreactor is a stainless steel chamber. In another embodiment, thebioreactor is a pre-sterilized plastic bag (e.g. Cellbag®, Wave Biotech,Bridgewater, N.J.). In other embodiment, the pre-sterilized plastic bagsare about 50 L to 3500 L bags.

Extraction and Purification of Nanoparticles Containing CoV Spike (S)Protein Antigens

After growth of the host cells, the protein may be harvested from thehost cells using detergents and purification protocols. Once the hostcells have grown for 48 to 96 hours, the cells are isolated from themedia and a detergent-containing solution is added to solubilize thecell membrane, releasing the protein in a detergent extract. TritonX-100 and TERGITOL® nonylphenol ethoxylate, also known as NP-9, are eachpreferred detergents for extraction. The detergent may be added to afinal concentration of about 0.1% to about 1.0%. For example, theconcentration may be about 0.1%, about 0.2%, about 0.3%, about 0.5%,about 0.7%, about 0.8%, or about 1.0%. The range may be about 0.1% toabout 0.3%. In aspects, the concentration is about 0.5%.

In other aspects, different first detergents may be used to isolate theprotein from the host cell. For example, the first detergent may beBis(polyethylene glycol bis[imidazoylcarbonyl]), nonoxynol-9,Bis(polyethylene glycol bis[imidazoyl carbonyl]), BRIJ® Polyethyleneglycol dodecyl ether 35, BRIJ® Polyethylene glycol (3) cetyl ether 56,BRIJ® alcohol ethoxylate 72, BRIJ® Polyoxyl 2 stearyl ether 76, BRIJ®polyethylene glycol monoolelyl ether 92V, BRIJ® Polyoxyethylene (10)oleyl ether 97, BRIJ® Polyethylene glycol hexadecyl ether 58P,CREMOPHOR® EL Macrogolglycerol ricinoleate, Decaethyleneglycolmonododecyl ether, N-Decanoyl-N-methylglucamine, n-Decylalpha-Dglucopyranoside,Decyl beta-D-maltopyranoside,n-Dodecanoyl-N-methylglucamide, nDodecyl alpha-D-maltoside, n-Dodecylbeta-D-maltoside, n-Dodecyl beta-D-maltoside,Heptaethylene glycolmonodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethyleneglycol monotetradecyl ether, n-Hexadecyl beta-D-maltoside, Hexaethyleneglycol monododecyl ether, Hexaethylene glycol monohexadecyl ether,Hexaethylene glycol monooctadecyl ether, Hexaethylene glycolmonotetradecyl ether, Igepal CA-630,Igepal CA-630,Methyl-6-0-(N-heptylcarbamoyl)-alpha-D-glucopyranoside,Nonaethyleneglycol monododecyl ether, N-Nonanoyl-N-methylglucamine,N-NonanoylN-methylglucamine, Octaethylene glycol monodecyl ether,Octaethylene glycolmonododecyl ether, Octaethylene glycol monohexadecylether, Octaethylene glycol monooctadecyl ether, Octaethylene glycolmonotetradecyl ether, Octyl-beta-D glucopyranoside, Pentaethylene glycolmonodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethyleneglycol monohexadecyl ether, Pentaethylene glycol monohexyl ether,Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctylether, Polyethylene glycol diglycidyl ether, Polyethylene glycol etherW-1, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate,Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether,Polyoxyethylene 40 stearate, Polyoxyethylene stearate, Polyoxyethylene 8stearate, Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene 25propylene glycol stearate, Saponin from Quillaja bark, SPAN® 20 sorbitanlaurate, SPAN® 40 sorbitan monopalmitate, SPAN® 60 sorbitan stearate,SPAN® 65 sorbitan tristearate, SPAN® 80 sorbitane monooleate, SPAN® 85sorbitane trioleate, TERGITOL® secondary alcohol ethoxylate Type15-S-12, TERGITOL® secondary alcohol ethoxylate Type 15-S-30, TERGITOL®secondary alcohol ethoxylate Type 15-S-5, TERGITOL® secondary alcoholethoxylate Type 15-S-7, TERGITOL® secondary alcohol ethoxylate Type15-S-9, TERGITOL® nonylphenol ethoxylate Type NP-10, TERGITOL®nonylphenol ethoxylate Type NP-4, TERGITOL® nonylphenol ethoxylate TypeNP-40, TERGITOL® nonylphenol ethoxylate Type NP-7, TERGITOL® nonylphenolethoxylate Type NP-9, TERGITOL® branched secondary alcohol ethoxylateType TMN-10, TERGITOL® branched secondary alcohol ethoxylate Type TMN-6,TRITON™ X-100 Polyethylene glycol tert-octylphenyl ether or combinationsthereof.

The nanoparticles may then be isolated from cellular debris usingcentrifugation. In embodiments, gradient centrifugation, such as usingcesium chloride, sucrose and iodixanol, may be used. Other techniquesmay be used as alternatives or in addition, such as standardpurification techniques including, e.g., ion exchange, affinity, and gelfiltration chromatography.

For example, the first column may be an ion exchange chromatographyresin, such as FRACTOGEL® EMD methacrylate based polymeric beads TMAE(EMD Millipore), the second column may be a lentil (Lens culinaris)lectin affinity resin, and the third column may be a cation exchangecolumn such as a FRACTOGEL® EMD methacrylate based polymeric beads S03(EMD Millipore) resin. In other aspects, the cation exchange column maybe an MMC column or a Nuvia C Prime column (Bio-Rad Laboratories, Inc).Preferably, the methods disclosed herein do not use a detergentextraction column; for example a hydrophobic interaction column. Such acolumn is often used to remove detergents during purification but maynegatively impact the methods disclosed here.

Detergent Exchange of Nanoparticles Containing CoV S PolypeptideAntigens

To form nanoparticles, the first detergent, used to extract the proteinfrom the host cell is substantially replaced with a second detergent toarrive at the nanoparticle structure. NP-9 is a preferred extractiondetergent. Typically, the nanoparticles do not contain detectable NP-9when measured by HPLC. The second detergent is typically selected fromthe group consisting of PS20, PS40, PS60, PS65, and PS80. Preferably,the second detergent is PS80.

In particular aspects, detergent exchange is performed using affinitychromatography to bind glycoproteins via their carbohydrate moiety. Forexample, the affinity chromatography may use a legume lectin column.Legume lectins are proteins originally identified in plants and found tointeract specifically and reversibly with carbohydrate residues. See,for example, Sharon and Lis, “Legume lectins—a large family ofhomologous proteins,” FASEB J. 1990 November; 4(14):3198-208; Liener,“The Lectins: Properties, Functions, and Applications in Biology andMedicine,” Elsevier, 2012. Suitable lectins include concanavalin A (conA), pea lectin, sainfoin lect, and lentil lectin. Lentil lectin is apreferred column for detergent exchange due to its binding properties.Lectin columns are commercially available; for example, Capto LentilLectin, is available from GE Healthcare. In certain aspects, the lentillectin column may use a recombinant lectin. At the molecular level, itis thought that the carbohydrate moieties bind to the lentil lectin,freeing the amino acids of the protein to coalesce around the detergentresulting in the formation of a detergent core providing nanoparticleshaving multiple copies of the antigen, e.g., glycoprotein oligomerswhich can be dimers, trimers, or tetramers anchored in the detergent. Inembodiments, the CoV S polypeptides form trimers. In embodiments, theCoV S polypeptide trimers are anchored in detergent. In embodiments,each CoV S polypeptide nanoparticle contains at least one trimerassociated with a non-ionic core.

The detergent, when incubated with the protein to form the nanoparticlesduring detergent exchange, may be present at up to about 0.1% (w/v)during early purifications steps and this amount is lowered to achievethe final nanoparticles having optimum stability. For example, thenon-ionic detergent (e.g., PS80) may be about 0.005% (v/v) to about 0.1%(v/v), for example, about 0.005% (v/v), about 0.006% (v/v), about 0.007%(v/v), about 0.008% (v/v), about 0.009% (v/v), about 0.01% (v/v), about0.015% (v/v), about 0.02% (v/v), about 0.025% (v/v), about 0.03% (v/v),about 0.035% (v/v), about 0.04% (v/v), about 0.045% (v/v), about 0.05%(v/v), about 0.055% (v/v), about 0.06% (v/v), about 0.065% (v/v), about0.07% (v/v), about 0.075% (v/v), about 0.08% (v/v), about 0.085% (v/v),about 0.09% (v/v), about 0.095% (v/v), or about 0.1% (v/v) PS80. Inembodiments, the nanoparticle contains about 0.03% to about 0.05% PS80.In embodiments, the nanoparticle contains about 0.01% (v/v) PS80.

In embodiments, purified CoV S polypeptides are dialyzed. Inembodiments, dialysis occurs after purification. In embodiments, the CoVS polypeptides are dialyzed in a solution comprising sodium phosphate,NaCl, and PS80. In embodiments, the dialysis solution comprising sodiumphosphate contains between about 5 mM and about 100 mM of sodiumphosphate, for example, about 5 mM, about 10 mM, about 15 mM, about 20mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM,about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100mM sodium phosphate. In embodiments, the pH of the solution comprisingsodium phosphate is about 6.5, about 6.6, about 6.7, about 6.8, about6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about7.5. In embodiments, the dialysis solution comprising sodium chloridecomprises about 50 mM NaCl to about 500 mM NaCl, for example, about 50mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM,about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM,about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM,about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM,about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM,about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM,about 360 mM, about 370 mM, about 380 mM, about 390 mM, about 400 mM,about 410 mM, about 420 mM, about 430 mM, about 440 mM, about 450 mM,about 460 mM, about 470 mM, about 480 mM, about 490 mM, or about 500 mMNaCl. In embodiments, the dialysis solution comprising PS80 comprisesabout 0.005% (v/v), about 0.006% (v/v), about 0.007% (v/v), about 0.008%(v/v), about 0.009% (v/v), about 0.01% (v/v), about 0.015% (v/v), about0.02% (v/v), about 0.025% (v/v), about 0.03% (v/v), about 0.035% (v/v),about 0.04% (v/v), about 0.045% (v/v), about 0.05% (v/v), about 0.055%(v/v), about 0.06% (v/v), about 0.065% (v/v), about 0.07% (v/v), about0.075% (v/v), about 0.08% (v/v), about 0.085% (v/v), about 0.09% (v/v),about 0.095% (v/v), or about 0.1% (v/v) PS80. In embodiments, thedialysis solution comprises about 25 mM sodium phosphate (pH 7.2), about300 mM NaCl, and about 0.01% (v/v) PS80.

Detergent exchange may be performed with proteins purified as discussedabove and purified, frozen for storage, and then thawed for detergentexchange.

Stability of compositions disclosed herein may be measured in a varietyof ways. In one approach, a peptide map may be prepared to determine theintegrity of the antigen protein after various treatments designed tostress the nanoparticles by mimicking harsh storage conditions. Thus, ameasure of stability is the relative abundance of antigen peptides in astressed sample compared to a control sample. For example, the stabilityof nanoparticles containing the CoV S polypeptides may be evaluated byexposing the nanoparticles to various pHs, proteases, salt, oxidizingagents, including but not limited to hydrogen peroxide, varioustemperatures, freeze/thaw cycles, and agitation. FIGS. 12A-B show thatBV2373 (SEQ ID NO: 87) and BV2365 (SEQ ID NO: 4) retain binding to hACE2under a variety of stress conditions. It is thought that the position ofthe glycoprotein anchored into the detergent core provides enhancedstability by reducing undesirable interactions. For example, theimproved protection against protease-based degradation may be achievedthrough a shielding effect whereby anchoring the glycoproteins into thecore at the molar ratios disclosed herein results in steric hindranceblocking protease access. Stability may also be measured by monitoringintact proteins. FIG. 33 and FIG. 34 compare nanoparticles containingCoV polypeptides having amino acid sequences of SEQ ID NOS: 109 and 87,respectively. FIG. 34 indicates that CoV polypeptides having an aminoacid sequence of SEQ ID NO: 87 show particularly good stability duringpurification. The polypeptide of FIG. 34 comprises a furin cleavage sitehaving an amino acid sequence of QQAQ (SEQ ID NO: 7).

Vaccine Compositions containing CoV S Polypeptide Antigens

The disclosure provides vaccine compositions comprising CoV Spolypeptides, for example, in a nanoparticle. In some aspects, thevaccine composition may contain nanoparticles with antigens from morethan one viral strain from the same species of virus. In anotherembodiment, the disclosures provide for a pharmaceutical pack or kitcomprising one or more containers filled with one or more of thecomponents of the vaccine compositions.

Compositions disclosed herein may be used either prophylactically ortherapeutically, but will typically be prophylactic. Accordingly, thedisclosure includes methods for treating or preventing infection. Themethods involve administering to the subject a therapeutic orprophylactic amount of the immunogenic compositions of the disclosure.Preferably, the pharmaceutical composition is a vaccine composition thatprovides a protective effect. In other aspects, the protective effectmay include amelioration of a symptom associated with infection in apercentage of the exposed population. For example, the composition mayprevent or reduce one or more virus disease symptoms selected from:fever fatigue, muscle pain, headache, sore throat, vomiting, diarrhea,rash, symptoms of impaired kidney and liver function, internal bleedingand external bleeding, compared to an untreated subject.

The nanoparticles may be formulated for administration as vaccines inthe presence of various excipients, buffers, and the like. For example,the vaccine compositions may contain sodium phosphate, sodium chloride,and/or histidine. Sodium phosphate may be present at about 10 mM toabout 50 mM, about 15 mM to about 25 mM, or about 25 mM; in particularcases, about 22 mM sodium phosphate is present. Histidine may be presentabout 0.1% (w/v), about 0.5% (w/v), about 0.7% (w/v), about 1% (w/v),about 1.5% (w/v), about 2% (w/v), or about 2.5% (w/v). Sodium chloride,when present, may be about 150 mM. In certain compositions, the sodiumchloride may be present in higher concentrations, for example from about200 mM to about 500 mM. In embodiments, the sodium chloride is presentin a high concentration, including but not limited to about 200 mM,about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, orabout 500 mM.

In embodiments, the nanoparticles described herein have improvedstability at certain pH levels. In embodiments, the nanoparticles arestable at slightly acidic pH levels. For example, the nanoparticles thatare stable at a slightly acidic pH, for example from pH 5.8 to pH 7.0.In embodiments, the nanoparticles and compositions containingnanoparticles may be stable at pHs ranging from about pH 5.8 to about pH7.0, including about pH 5.9 to about pH 6.8, about pH 6.0 to about pH6.5, about pH 6.1 to about pH 6.4, about pH 6.1 to about pH 6.3, orabout pH 6.2. In embodiments, the nanoparticles and compositionsdescribed herein are stabile at neutral pHs, including from about pH 7.0to about pH 7.4. In embodiments, the nanoparticles and compositionsdescribed herein are stable at slightly alkaline pHs, for example fromabout pH 7.0 to about pH 8.5, from about pH 7.0 to about pH 8.0, or fromabout pH 7.0 to about pH 7.5, including all values and ranges inbetween.

Adjuvants

In certain embodiments, the compositions disclosed herein may becombined with one or more adjuvants to enhance an immune response. Inother embodiments, the compositions are prepared without adjuvants, andare thus available to be administered as adjuvant-free compositions.Advantageously, adjuvant-free compositions disclosed herein may provideprotective immune responses when administered as a single dose.Alum-free compositions that induce robust immune responses areespecially useful in adults about 60 and older.

Aluminum-Based Adjuvants

In embodiments, the adjuvant may be alum (e.g. AlPO₄ or Al(OH)₃).Typically, the nanoparticle is substantially bound to the alum. Forexample, the nanoparticle may be at least 80% bound, at least 85% bound,at least 90% bound or at least 95% bound to the alum. Often, thenanoparticle is 92% to 97% bound to the alum in a composition. Theamount of alum is present per dose is typically in a range between about400 μg to about 1250 μg. For example, the alum may be present in a perdose amount of about 300 μg to about 900 μg, about 400 μg to about 800μg, about 500 μg to about 700 μg, about 400 μg to about 600 μg, or about400 μg to about 500 μg. Typically, the alum is present at about 400 μgfor a dose of 120 μg of the protein nanoparticle.

Saponin Adjuvants

Adjuvants containing saponin may also be combined with the immunogensdisclosed herein. Saponins are glycosides derived from the bark of theQuillaja saponaria Molina tree. Typically, saponin is prepared using amulti-step purification process resulting in multiple fractions. Asused, herein, the term “a saponin fraction from Quillaja saponariaMolina” is used generically to describe a semi-purified or definedsaponin fraction of Quillaja saponaria or a substantially pure fractionthereof.

Saponin Fractions

Several approaches for producing saponin fractions are suitable.Fractions A, B, and C are described in U.S. Pat. No. 6,352,697 and maybe prepared as follows. A lipophilic fraction from Quil A, a crudeaqueous Quillaja saponaria Molina extract, is separated bychromatography and eluted with 70% acetonitrile in water to recover thelipophilic fraction. This lipophilic fraction is then separated bysemi-preparative HPLC with elution using a gradient of from 25% to 60%acetonitrile in acidic water. The fraction referred to herein as“Fraction A” or “QH-A” is, or corresponds to, the fraction, which iseluted at approximately 39% acetonitrile. The fraction referred toherein as “Fraction B” or “QH-B” is, or corresponds to, the fraction,which is eluted at approximately 47% acetonitrile. The fraction referredto herein as “Fraction C” or “QH-C” is, or corresponds to, the fraction,which is eluted at approximately 49% acetonitrile. Additionalinformation regarding purification of Fractions is found in U.S. Pat.No. 5,057,540. When prepared as described herein, Fractions A, B and Cof Quillaja saponaria Molina each represent groups or families ofchemically closely related molecules with definable properties. Thechromatographic conditions under which they are obtained are such thatthe batch-to-batch reproducibility in terms of elution profile andbiological activity is highly consistent.

Other saponin fractions have been described. Fractions B3, B4 and B4bare described in EP 0436620. Fractions QA1-QA22 are described EP03632279B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria Molina Spikoside(lsconova AB, Ultunaallén 2B, 756 51 Uppsala, Sweden). Fractions QA-1,QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11, QA-12,QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21, and QA-22of EP 0 3632 279 B2, especially QA-7, QA-17, QA-18, and QA-21 may beused. They are obtained as described in EP 0 3632 279 B2, especially atpage 6 and in Example 1 on page 8 and 9.

The saponin fractions described herein and used for forming adjuvantsare often substantially pure fractions; that is, the fractions aresubstantially free of the presence of contamination from othermaterials. In particular aspects, a substantially pure saponin fractionmay contain up to 40% by weight, up to 30% by weight, up to 25% byweight, up to 20% by weight, up to 15% by weight, up to 10% by weight,up to 7% by weight, up to 5% by weight, up to 2% by weight, up to 1% byweight, up to 0.5% by weight, or up to 0.1% by weight of other compoundssuch as other saponins or other adjuvant materials.

ISCOM Structures

Saponin fractions may be administered in the form of a cage-likeparticle referred to as an ISCOM (Immune Stimulating COMplex). ISCOMsmay be prepared as described in EP0109942B1, EP0242380B1 and EP0180546B1. In particular embodiments a transport and/or a passenger antigen maybe used, as described in EP 9600647-3 (PCT/SE97/00289).

Matrix Adjuvants

In embodiments, the ISCOM is an ISCOM matrix complex. An ISCOM matrixcomplex comprises at least one saponin fraction and a lipid. The lipidis at least a sterol, such as cholesterol. In particular aspects, theISCOM matrix complex also contains a phospholipid. The ISCOM matrixcomplexes may also contain one or more other immunomodulatory(adjuvant-active) substances, not necessarily a glycoside, and may beproduced as described in EP0436620B1, which is incorporated by referencein its entirety herein.

In other aspects, the ISCOM is an ISCOM complex. An ISCOM complexcontains at least one saponin, at least one lipid, and at least one kindof antigen or epitope. The ISCOM complex contains antigen associated bydetergent treatment such that that a portion of the antigen integratesinto the particle. In contrast, ISCOM matrix is formulated as anadmixture with antigen and the association between ISCOM matrixparticles and antigen is mediated by electrostatic and/or hydrophobicinteractions.

According to one embodiment, the saponin fraction integrated into anISCOM matrix complex or an ISCOM complex, or at least one additionaladjuvant, which also is integrated into the ISCOM or ISCOM matrixcomplex or mixed therewith, is selected from fraction A, fraction B, orfraction C of Quillaja saponaria, a semipurified preparation of Quillajasaponaria, a purified preparation of Quillaja saponaria, or any purifiedsub-fraction e.g., QA 1-21.

In particular aspects, each ISCOM particle may contain at least twosaponin fractions. Any combinations of weight % of different saponinfractions may be used. Any combination of weight % of any two fractionsmay be used. For example, the particle may contain any weight % offraction A and any weight % of another saponin fraction, such as a crudesaponin fraction or fraction C, respectively. Accordingly, in particularaspects, each ISCOM matrix particle or each ISCOM complex particle maycontain from 0.1 to 99.9 by weight, 5 to 95% by weight, 10 to 90% byweight 15 to 85% by weight, 20 to 80% by weight, 25 to 75% by weight, 30to 70% by weight, 35 to 65% by weight, 40 to 60% by weight, 45 to 55% byweight, 40 to 60% by weight, or 50% by weight of one saponin fraction,e.g. fraction A and the rest up to 100% in each case of another saponine.g. any crude fraction or any other faction e.g. fraction C. The weightis calculated as the total weight of the saponin fractions. Examples ofISCOM matrix complex and ISCOM complex adjuvants are disclosed in U.SPublished Application No. 2013/0129770, which is incorporated byreference in its entirety herein.

In particular embodiments, the ISCOM matrix or ISCOM complex comprisesfrom 5-99% by weight of one fraction, e.g. fraction A and the rest up to100% of weight of another fraction e.g. a crude saponin fraction orfraction C. The weight is calculated as the total weight of the saponinfractions.

In another embodiment, the ISCOM matrix or ISCOM complex comprises from40% to 99% by weight of one fraction, e.g. fraction A and from 1% to 60%by weight of another fraction, e.g. a crude saponin fraction or fractionC. The weight is calculated as the total weight of the saponinfractions.

In yet another embodiment, the ISCOM matrix or ISCOM complex comprisesfrom 70% to 95% by weight of one fraction e.g., fraction A, and from 30%to 5% by weight of another fraction, e.g., a crude saponin fraction, orfraction C. The weight is calculated as the total weight of the saponinfractions. In other embodiments, the saponin fraction from Quillajasaponaria Molina is selected from any one of QA 1-21.

In addition to particles containing mixtures of saponin fractions, ISCOMmatrix particles and ISCOM complex particles may each be formed usingonly one saponin fraction. Compositions disclosed herein may containmultiple particles wherein each particle contains only one saponinfraction. That is, certain compositions may contain one or moredifferent types of ISCOM-matrix complexes particles and/or one or moredifferent types of ISCOM complexes particles, where each individualparticle contains one saponin fraction from Quillaja saponaria Molina,wherein the saponin fraction in one complex is different from thesaponin fraction in the other complex particles.

In particular aspects, one type of saponin fraction or a crude saponinfraction may be integrated into one ISCOM matrix complex or particle andanother type of substantially pure saponin fraction, or a crude saponinfraction, may be integrated into another ISCOM matrix complex orparticle. A composition or vaccine may comprise at least two types ofcomplexes or particles each type having one type of saponins integratedinto physically different particles.

In the compositions, mixtures of ISCOM matrix complex particles and/orISCOM complex particles may be used in which one saponin fractionQuillaja saponaria Molina and another saponin fraction Quillajasaponaria Molina are separately incorporated into different ISCOM matrixcomplex particles and/or ISCOM complex particles.

The ISCOM matrix or ISCOM complex particles, which each have one saponinfraction, may be present in composition at any combination of weight %.In particular aspects, a composition may contain 0.1% to 99.9% byweight, 5% to 95% by weight, 10% to 90% by weight, 15% to 85% by weight,20% to 80% by weight, 25% to 75% by weight, 30% to 70% by weight, 35% to65% by weight, 40% to 60% by weight, 45% to 55% by weight, 40 to 60% byweight, or 50% by weight, of an ISCOM matrix or complex containing afirst saponin fraction with the remaining portion made up by an ISCOMmatrix or complex containing a different saponin fraction. In someaspects, the remaining portion is one or more ISCOM matrix or complexeswhere each matrix or complex particle contains only one saponinfraction. In other aspects, the ISCOM matrix or complex particles maycontain more than one saponin fraction.

In particular compositions, the only saponin fraction in a first ISCOMmatrix or ISCOM complex particle is Fraction A and the only saponinfraction in a second ISCOM matrix or ISCOM complex particle is FractionC.

Preferred compositions comprise a first ISCOM matrix containing FractionA and a second ISCOM matrix containing Fraction C, wherein the FractionA ISCOM matrix constitutes about 70% per weight of the total saponinadjuvant, and the Fraction C ISCOM matrix constitutes about 30% perweight of the total saponin adjuvant. In another preferred composition,the Fraction A ISCOM matrix constitutes about 85% per weight of thetotal saponin adjuvant, and the Fraction C ISCOM matrix constitutesabout 15% per weight of the total saponin adjuvant. Thus, in certaincompositions, the Fraction A ISCOM matrix is present in a range of about70% to about 85%, and Fraction C ISCOM matrix is present in a range ofabout 15% to about 30%, of the total weight amount of saponin adjuvantin the composition. In embodiments, the Fraction A ISCOM matrix accountsfor 50-96% by weight and Fraction C ISCOM matrix accounts for theremainder, respectively, of the sums of the weights of Fraction A ISCOMmatrix and Fraction C ISCOM in the adjuvant. In a particularly preferredcomposition, referred to herein as MATRIX-M™, the Fraction A ISCOMmatrix is present at about 85% and Fraction C ISCOM matrix is present atabout 15% of the total weight amount of saponin adjuvant in thecomposition. MATRIX-M™ may be referred to interchangeably as Matrix-M1.

Exemplary QS-7 and QS-21 fractions, their production and their use isdescribed in U.S. Pat. Nos. 5,057,540; 6,231,859; 6,352,697; 6,524,584;6,846,489; 7,776,343, and 8,173,141, which are incorporated by referenceherein.

In some, compositions other adjuvants may be used in addition or as analternative. The inclusion of any adjuvant described in Vogel et al., “ACompendium of Vaccine Adjuvants and Excipients (2nd Edition),” hereinincorporated by reference in its entirety for all purposes, isenvisioned within the scope of this disclosure. Other adjuvants includecomplete Freund's adjuvant (a non-specific stimulator of the immuneresponse containing killed Mycobacterium tuberculosis), incompleteFreund's adjuvants and aluminum hydroxide adjuvant. Other adjuvantscomprise GMCSP, BCG, MDP compounds, such as thur-MDP and nor-MDP, CGP(MTP-PE), lipid A, and monophosphoryl lipid A (MPL), MF-59, RIBI, whichcontains three components extracted from bacteria, MPL, trehalosedimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/TWEEN®polysorbate 80 emulsion. In embodiments, the adjuvant may be apaucilamellar lipid vesicle; for example, NOVASOMES®. NOVASOMES® arepaucilamellar nonphospholipid vesicles ranging from about 100 nm toabout 500 nm. They comprise BRIJ® alcohol ethoxylate 72, cholesterol,oleic acid and squalene. NOVASOMES® have been shown to be an effectiveadjuvant (see, U.S. Pat. Nos. 5,629,021, 6,387,373, and 4,911,928.

Administration and Dosage

In embodiments, the disclosure provides a method for eliciting an immuneresponse against one or more coronaviruses. In embodiments, the responseis against one or more of the SARS-CoV-2 virus, MERS, and SARS. Themethod involves administering an immunologically effective amount of acomposition containing a nanoparticle or containing a recombinant CoVSpike (S) polypeptide to a subject. Advantageously, the proteinsdisclosed herein induce one or more of particularly usefulanti-coronavirus responses.

In embodiments, the nanoparticles or CoV S polypeptides are administeredwith an adjuvant. In other aspects, the nanoparticles or CoV Spolypeptides are administered without an adjuvant. In some aspects, theadjuvant may be bound to the nanoparticle, such as by a non-covalentinteraction. In other aspects, the adjuvant is co-administered with thenanoparticle but the adjuvant and nanoparticle do not interactsubstantially.

In embodiments, the nanoparticles may be used for the prevention and/ortreatment of one or more of a SARS-CoV-2 infection, a SARS infection, ora MERS infection. Thus, the disclosure provides a method for elicitingan immune response against one or more of the SARS-CoV-2 virus, MERS,and SARS. The method involves administering an immunologically effectiveamount of a composition containing a nanoparticle or a CoV S polypeptideto a subject. Advantageously, the proteins disclosed herein induceparticularly useful anti-coronavirus responses.

Compositions disclosed herein may be administered via a systemic routeor a mucosal route or a transdermal route or directly into a specifictissue. As used herein, the term “systemic administration” includesparenteral routes of administration. In particular, parenteraladministration includes subcutaneous, intraperitoneal, intravenous,intraarterial, intramuscular, or intrasternal injection, intravenous, orkidney dialytic infusion techniques. Typically, the systemic, parenteraladministration is intramuscular injection. As used herein, the term“mucosal administration” includes oral, intranasal, intravaginal,intra-rectal, intra-tracheal, intestinal and ophthalmic administration.Preferably, administration is intramuscular.

Compositions may be administered on a single dose schedule or a multipledose schedule. Multiple doses may be used in a primary immunizationschedule or in a booster immunization schedule. In a multiple doseschedule the various doses may be given by the same or different routese.g., a parenteral prime and mucosal boost, a mucosal prime andparenteral boost, etc. In some aspects, a follow-on boost dose isadministered about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,or about 6 weeks after the prior dose. In embodiments, the follow-onboost dose is administered 3 weeks after administration of the priordose. In embodiments, the first dose is administered at day 0, and theboost dose is administered at day 21. In embodiments, the first dose isadministered at day 0, and the boost dose is administered at day 28.

In embodiments, the dose, as measured in μg, may be the total weight ofthe dose including the solute, or the weight of the CoV S polypeptidenanoparticles, or the weight of the CoV S polypeptide. Dose is measuredusing protein concentration assay either A280 or ELISA.

The dose of antigen, including for pediatric administration, may be inthe range of about 5 μg to about 25 μg, about 1 μg to about 300 μg,about 90 μg to about 270 μg, about 100 μg to about 160 μg, about 110 μgto about 150 μg, about 120 μg to about 140 μg, or about 140 μg to about160 μg. In embodiments, the dose is about 120 μg, administered withalum. In some aspects, a pediatric dose may be in the range of about 1μg to about 90 μg. In embodiments, the dose of CoV Spike (S) polypeptideis about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg,about 18 μg, about 19 μg, about 20 μg, about 21, about 22, about 23,about 24, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29μg, about 30 μg, about 40 μg, about 50, about 60, about 70, about 80,about 90 about 100 μg, about 110 μg, about 120 μg, about 130 μg, about140 μg, about 150 μg, about 160 μg, about 170 μg, about 180 μg, about190 μg, about 200 μg, about 210 μg, about 220 μg, about 230 μg, about240 μg, about 250 μg, about 260 μg, about 270 μg, about 280 μg, about290 μg, or about 300 μg, including all values and ranges in between. Inembodiments, the dose of CoV S polypeptide is 5 μg. In embodiments, thedose of CoV S polypeptide is 25 μg.

Certain populations may be administered with or without adjuvants. Incertain aspects, compositions may be free of added adjuvant. In suchcircumstances, the dose may be increased by about 10%.

In embodiments, the dose of the adjuvant administered with anon-naturally occurring CoV S polypeptide is from about 1 μg to about100 μg, for example, about 1 μg, about 2 μg, about 3 μg, about 4 μg,about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg,about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21,about 22, about 23, about 24, about 25 μg, about 26 μg, about 27 μg,about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg,about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg,about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg,about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg,about 72 μg, about 73 μg, about 74 μg, about 75 μg, about 76 μg, about77 μg, about 78 μg, about 79 μg, about 80 μg, about 81 μg, about 82 μg,about 83 μg, about 84 μg, about 85 μg, about 86 μg, about 87 μg, about88 μg, about 89 μg, about 90 μg, about 91 μg, about 92 μg, about 93 μg,about 94 μg, about 95 μg, about 96 μg, about 97 μg, about 98 μg, about99 μg, or about 100 μg of adjuvant. In embodiments, the dose of adjuvantis about 50 μg. In embodiments, the adjuvant is a saponin adjuvant,e.g., MATRIX-M™.

In embodiments, the dose is administered in a volume of about 0.1 mL toabout 1.5 mL, for example, about 0.1 mL, about 0.2 mL, about 0.25 mL,about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL,about 0.8 mL, about 0.9 mL, about 1.0 mL, about 1.1 mL, about 1.2 mL,about 1.3 mL, about 1.4 mL, or about 1.5 mL. In embodiments, the dose isadministered in a volume of 0.25 mL. In embodiments, the dose isadministered in a volume of 0.5 mL. In embodiments, the dose isadministered in a volume of 0.6 mL.

In particular embodiments for a vaccine against MERS, SARS, or theSARS-CoV-2 coronavirus, the dose may comprise a CoV S polypeptideconcentration of about 1 μg/mL to about 50 μg/mL, 10 μg/mL to about 100μg/mL, about 10 μg/mL to about 50 μg/mL, about 175 μg/mL to about 325μg/mL, about 200 μg/mL to about 300 μg/mL, about 220 μg/mL to about 280μg/mL, or about 240 μg/mL to about 260 μg/mL.

In another embodiment, the disclosure provides a method of formulating avaccine composition that induces immunity to an infection or at leastone disease symptom thereof to a mammal, comprising adding to thecomposition an effective dose of a nanoparticle or a CoV S polypeptide.The disclosed CoV S polypeptides and nanoparticles are useful forpreparing compositions that stimulate an immune response that confersimmunity or substantial immunity to infectious agents. Thus, in oneembodiment, the disclosure provides a method of inducing immunity toinfections or at least one disease symptom thereof in a subject,comprising administering at least one effective dose of a nanoparticleand/or a CoV S polypeptide.

In embodiments, the CoV S polypeptides or nanoparticles comprising thesame are administered in combination with an additional immunogeniccomposition. In embodiments, the additional immunogenic compositioninduces an immune response against SARS-CoV-2. In embodiments, theadditional immunogenic composition is administered within about 1minute, about 5 minutes, about 10 minutes, about 20 minutes, about 30minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, about 1 day, about 2days, about 3 days, about 4 days, about 5 days, about 6 days, about 7days, about 8 days, about 9 days, about 10 days, about 11 days, about 12days, about 13 days, about 14 days, about 15 days, about 16 days, about17 days, about 18 days, about 19 days, about 20 days, about 21 days,about 22 days, about 23 days, about 24 days, about 25 days, about 26days, about 27 days, about 28 days, about 29 days, about 30 days, orabout 31 days of the disclosed CoV S polypeptides or nanoparticlescomprising the same. In embodiments, the additional composition isadministered with a first dose of a composition comprising a CoV Spolypeptide or nanoparticle comprising the same. In embodiments, theadditional composition is administered with a boost dose of acomposition comprising a CoV S polypeptide or nanoparticle comprisingthe same.

In embodiments, the additional immunogenic composition comprises an mRNAencoding a SARS-Cov-2 Spike glycoprotein, a plasmid DNA encoding aSARS-Cov-2 Spike glycoprotein, an viral vector encoding a SARS-Cov-2Spike glycoprotein, or an inactivated SARS-CoV-2 virus.

In embodiments, the additional immunogenic composition comprises mRNAthat encodes for a CoV S polypeptide. In embodiments, the mRNA encodesfor a CoV S polypeptide comprising proline substitutions at positions986 and 987 of SEQ ID NO: 1. In embodiments, the mRNA encodes for a CoVS polypeptide comprising an intact furin cleavage site. In embodiments,the mRNA encodes for a CoV S polypeptide comprising prolinesubstitutions at positions 986 and 987 of SEQ ID NO: 1 and an intactfurin cleavage site. In embodiments, the mRNA encodes for a CoV Spolypeptide comprising proline substitutions at positions 986 and 987 ofSEQ ID NO: 1 and an inactive furin cleavage site. In embodiments, themRNA encodes for a CoV S polypeptide having an amino acid sequence ofSEQ ID NO: 87. In embodiments, the mRNA encoding for a CoV S polypeptideis encapsulated in a lipid nanoparticle. An exemplary immunogeniccomposition comprising mRNA that encodes for a CoV S polypeptide isdescribed in Jackson et al. N. Eng. J. Med. 2020. An mRNA Vaccineagainst SARS-CoV-2-preliminary report, which is incorporated byreference in its entirety herein. In embodiments, the compositioncomprising mRNA that encodes for a CoV S polypeptide is administered ata dose of 25 μg, 100 μg, or 250 μg.

In embodiments, the additional immunogenic composition comprises anadenovirus vector encoding for a CoV S polypeptide. In embodiments, theAAV vector encodes for a wild-type CoV S polypeptide. In embodiments,the AAV vector encodes for a CoV S polypeptide comprising prolinesubstitutions at positions 986 and 987 of SEQ ID NO: 1 and an intactfurin cleavage site. In embodiments, the AAV vector encodes for a CoV Spolypeptide comprising proline substitutions at positions 986 and 987 ofSEQ ID NO: 1 and an inactive furin cleavage site. In embodiments, theAAV vector encodes for a CoV S polypeptide having an amino acid sequenceof SEQ ID NO: 87. The following publications describe immunogeniccompositions comprising an adenovirus vector encoding for a CoV Spolypeptide, each of which is incorporated by reference in its entiretyherein: van Doremalen N. et al. A single dose of ChAdOx1 MERS providesprotective immunity in rhesus macaques. Science Advances, 2020; vanDoremalen N. et al. ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2pneumonia in rhesus macaques. bioRxiv, (2020).

In embodiments, the additional immunogenic composition comprisesdeoxyribonucleic acid (DNA). In embodiments, the additional immunogeniccomposition comprises plasmid DNA. In embodiments, the plasmid DNAencodes for a CoV S polypeptide. In embodiments, the DNA encodes for aCoV S polypeptide comprising proline substitutions at positions 986 and987 of SEQ ID NO: 1 and an intact furin cleavage site. In embodiments,the DNA encodes for a CoV S polypeptide comprising proline substitutionsat positions 986 and 987 of SEQ ID NO: 1 and an inactive furin cleavagesite. In embodiments, the DNA encodes for a CoV S polypeptide having anamino acid sequence of SEQ ID NO: 87.

In embodiments, the additional immunogenic composition comprises aninactivated virus vaccine.

In embodiments, the CoV S proteins or nanoparticles comprising CoV Sproteins are useful for preparing immunogenic compositions to stimulatean immune response that confers immunity or substantial immunity to oneor more of MERS, SARS, and SARS-CoV-2. Both mucosal and cellularimmunity may contribute to immunity to infection and disease. Antibodiessecreted locally in the upper respiratory tract are a major factor inresistance to natural infection. Secretory immunoglobulin A (sIgA) isinvolved in protection of the upper respiratory tract and serum IgG inprotection of the lower respiratory tract. The immune response inducedby an infection protects against reinfection with the same virus or anantigenically similar viral strain. The antibodies produced in a hostafter immunization with the nanoparticles disclosed herein can also beadministered to others, thereby providing passive administration in thesubject.

In embodiments, the present disclosure provides a method of producingone or more of high affinity anti-MERS-CoV, anti-SARS-CoV, andanti-SARS-CoV-2 virus antibodies. The high affinity antibodies producedby immunization with the nanoparticles disclosed herein are produced byadministering an immunogenic composition comprising an S CoV polypeptideor a nanoparticle comprising an S CoV polypeptide to an animal,collecting the serum and/or plasma from the animal, and purifying theantibody from the serum/and or plasma. In one embodiment, the animal isa human. In embodiments, the animal is a chicken, mouse, guinea pig,rat, rabbit, goat, human, horse, sheep, or cow. In one embodiment, theanimal is bovine or equine. In another embodiment, the bovine or equineanimal is transgenic. In yet a further embodiment, the transgenic bovineor equine animal produces human antibodies. In embodiments, the animalproduces monoclonal antibodies. In embodiments, the animal producespolyclonal antibodies. In one embodiment, the method further comprisesadministration of an adjuvant or immune stimulating compound. In afurther embodiment, the purified high affinity antibody is administeredto a human subject. In one embodiment, the human subject is at risk forinfection with one or more of MERS, SARS, and SARS-CoV-2.

All patents, patent applications, references, and journal articles citedin this disclosure are expressly incorporated herein by reference intheir entireties for all purposes.

EXAMPLES Example 1 Expression and Purification of Coronavirus Spike (S)Polypeptide Nanoparticles

The native coronavirus Spike (S) polypeptide (SEQ ID NO: 1 and SEQ IDNO:2) and CoV Spike polypeptides which have amino acid sequencescorresponding to SEQ ID NOS: 3, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63,65, 67, 73, 75, 78, 79, 82, 83, 85, 87, 106, 108, and 89 have beenexpressed in a baculovirus expression system and recombinant plaquesexpressing the coronavirus Spike (S) polypeptides were picked andconfirmed. In each case the signal peptide is SEQ ID NO: 5. FIG. 4 andFIG. 9 show successful purification of the CoV Spike polypeptidesBV2364, BV2365, BV2366, BV2367, BV2368, BV2369, BV2373, BV2374, andBV2375. Table 2 shows the sequence characteristics of the aforementionedCoV Spike polypeptides.

TABLE 2 Selected CoV Spike Polypeptides SEQ ID CoV S polypeptideModification NO. BV2364 Deleted N-Terminal Domain 48 BV2365 Inactivefurin cleavage site 4 BV2361/BV2366 Wild-type 2 BV2367 Deletion of aminoacids 676- 63 685, inactive furin cleavage site BV2368 Deletion of aminoacids 702- 65 711, inactive furin cleavage site BV2369 Deletion of aminoacids 806- 67 815, inactive furin cleavage site BV2373, formulated intoa Inactive furin cleavage site, 87 composition referred to herein K973Pmutation, V974P as “NVX-CoV2373” mutation BV2374 K973P mutation, V974P85 mutation BV2374 Inactive furin cleavage site 58 and His-tag BV2384Inactive furin cleavage site 110 (GSAS), K973P, V974P mutation

The wild-type BV2361 protein (SEQ ID NO: 2) binds to humanangiotensin-converting enzyme 2 precursor (hACE2). Bio-layerinterferometry and ELISA were performed to assess binding of the CoV Spolypeptides.

Bio-Layer Interferometry (BLI):

The BLI experiments were performed using an Octet QK384 system (PallForte Bio, Fremont, Calif.). His-tagged human ACE2 (2 μg mL-1) wasimmobilized on nickel-charged Ni-NTA biosensor tips. After baseline,SARS-CoV-2 S protein containing samples were 2-fold serially diluted andwere allowed to associate for 600 seconds followed by dissociation foran additional 900 sec. Data was analyzed with Octet software HT 101:1global curve fit.

The CoV S polypeptides BV2361, BV2365, BV2369, BV2365, BV2373, BV2374retain the ability to bind to hACE2 (FIG. 5, FIGS. 11A-C). Dissociationkinetics showed that the S-proteins remained tightly bound as evident byminimal or no dissociation over 900 seconds of observation in theabsence of fluid phase S protein (FIGS. 11A-C).

Furthermore, binding is specific. The wild-type CoV S protein, BV2361and the CoV S polypeptides BV2365 and BV2373 do not bind the MERS-CoVreceptor, dipeptidyl peptidase IV (DPP4). Additionally, the MERS Sprotein does not bind to human angiotensin-converting enzyme 2 precursor(hACE2) (FIG. 6 and FIGS. 11D-F).

ELISA

The specificity of the CoV S polypeptides for hACE2 was confirmed byELISA. Ninety-six well plates were coated with 100 μL SARS-CoV-2 spikeprotein (2 μg/mL) overnight at 4° C. Plates were washed with phosphatebuffered saline with 0.05% Tween (PBS-T) buffer and blocked with TBSStartblock blocking buffer (ThermoFisher, Scientific). His-tagged hACE2and hDPP4 receptors were 3-fold serially diluted (5-0.0001 μg mL-1) andadded to coated wells for 2 hours at room temperature. The plates werewashed with PBS-T. Optimally diluted horseradish peroxidase (HRP)conjugated anti-histidine was added and color developed by addition ofand 3,3′,5,5′-tetramethylbenzidine peroxidase substrate (TMB, T0440-IL,Sigma, St. Louis, Mo., USA). Plates were read at an OD of 450 nm with aSpectraMax Plus plate reader (Molecular Devices, Sunnyvale, Calif., USA)and data analyzed with SoftMax software. EC50 values were calculated by4-parameter fitting using GraphPad Prism 7.05 software.

The ELISA results showed that the wild-type CoV S polypeptide (BV2361),BV2365, and BV2373 proteins specifically bound hACE2 but failed to bindthe hDPP-4 receptor used by MERS-CoV (IC₅₀>5000 ng mL-1). The wild-typeCoV S polypeptide and BV2365 bound to hACE2 with similar affinity(IC₅₀=36-38 ng/mL), while BV2373 attained 50% saturation of hACE2binding at 2-fold lower concentration (IC₅₀=18 ng/mL) (FIG. 7, FIGS.11D-F).

Protein and Nanoparticle Production

The recombinant virus is amplified by infection of Sf9 insect cells. Aculture of insect cells is infected at ˜3 MOI (Multiplicity ofinfection=virus ffu or pfu/cell) with baculovirus. The culture andsupernatant is harvested 48-72 hrs post-infection. The crude cellharvest, approximately 30 mL, is clarified by centrifugation for 15minutes at approximately 800×g. The resulting crude cell harvestscontaining the coronavirus Spike (S) protein are purified asnanoparticles as described below.

To produce nanoparticles, non-ionic surfactant TERGITOL® nonylphenolethoxylate NP-9 is used in the membrane protein extraction protocol.Crude extraction is further purified by passing through anion exchangechromatography, lentil lectin affinity/HIC and cation exchangechromatography. The washed cells are lysed by detergent treatment andthen subjected to low pH treatment which leads to precipitation of BVand Sf9 host cell DNA and protein. The neutralized low pH treatmentlysate is clarified and further purified on anion exchange and affinitychromatography before a second low pH treatment is performed.

Affinity chromatography is used to remove 519/BV proteins, DNA and NP-9,as well as to concentrate the coronavirus Spike (S) protein. Briefly,lentil lectin is a metalloprotein containing calcium and manganese,which reversibly binds polysaccharides and glycosylated proteinscontaining glucose or mannose. The coronavirus Spike (S)protein-containing anion exchange flow through fraction is loaded ontothe lentil lectin affinity chromatography resin (Capto Lentil Lectin, GEHealthcare). The glycosylated coronavirus Spike (S) protein isselectively bound to the resin while non-glycosylated proteins and DNAare removed in the column flow through. Weakly bound glycoproteins areremoved by buffers containing high salt and low molar concentration ofmethyl alpha-D-mannopyranoside (MMP).

The column washes are also used to detergent exchange the NP-9 detergentwith the surfactant polysorbate 80 (PS80). The coronavirus Spike (S)polypeptides are eluted in nanoparticle structure from the lentil lectincolumn with a high concentration of MMP. After elution, the coronavirusSpike (S) protein trimers are assembled into nanoparticles composed ofcoronavirus Spike (S) protein trimers and PS80 contained in a detergentcore.

Example 2 Immunogenicity of Coronavirus Spike (S) PolypeptideNanoparticle Vaccines in Mice

The coronavirus Spike (S) protein composition comprising a CoV Spolypeptide of SEQ ID NO: 87 (also called “BV2373”) as described inExample 1 was evaluated for immunogenicity and toxicity in a murinemodel, using female BALB/c mice (7-9 weeks old; Harlan LaboratoriesInc., Frederick, Md.). The compositions were evaluated in the presenceand in the absence of a saponin adjuvant, e.g., MATRIX-M™. Compositionscontaining MATRIX-M™ contained 5 μg of MATRIX-M™. Vaccines containingcoronavirus Spike (S) polypeptide at various doses, including 0.01 μg,0.1 μg, 1 μg, and 10 μg, were administered intramuscularly as a singledose (also referred to as a single priming dose) (study day 14) or astwo doses (also referred to as a prime/boost regimen) spaced 14-daysapart (study day 0 and 14). A placebo group served as a non-immunizedcontrol. Serum was collected for analysis on study days—1, 13, 21, and28. Vaccinated and control animals were intranasally challenged withSARS-CoV-2 42 days following one (a single dose) or two (two doses)immunizations.

Vaccine Immunogenicity

Animals immunized with a single priming dose of 0.1-10 μg BV2373 andMATRIX-M™ had elevated anti-S IgG titers that were detected 21-28 daysafter a single immunization (FIG. 13B). Mice immunized with a 10 μg doseof BV2373 and MATRIX-M™ produced antibodies that blocked hACE2 receptorbinding to the CoV S protein and virus neutralizing antibodies that weredetected 21-28 days after a single priming dose (FIG. 14 and FIG. 15).Animals immunized with the prime/boost regimen (two doses) hadsignificantly elevated anti-S IgG titers that were detected 7-16 daysfollowing the booster immunization across all dose levels (FIG. 13A).Animals immunized with BV2373 (1 μg and 10 μg) and MATRIX-M™ had similarhigh anti-S IgG titers following immunization (GMT=139,000 and 84,000,respectively). Mice immunized with BV2373 (0.1 μg, 1 μg, or 10 μg) andMATRIX-M™ had significantly (p<0.05 and p<0.0001) higher anti-S IgGtiters compared to mice immunized with 10 μg BV2373 without adjuvant(FIG. 13A). These results indicate the potential for 10- to 100-folddose sparing provided by the MATRIX-M™ adjuvant. Furthermore,immunization with two doses of BV2373 and MATRIX-M™ elicited high titerantibodies that blocked hACE2 receptor binding to S-protein(IC50=218-1642) and neutralized the cytopathic effect (CPE) ofSARS-CoV-2 on Vero E6 cells (100% blocking of CPE=7680-20,000) acrossall dose levels (FIG. 14 and FIG. 15).

SARS CoV-2 Challenge

To evaluate the induction of protective immunity, immunized mice werechallenged with SARS-CoV-2. Since mice do not support replication of thewild-type SARS-CoV-2 virus, on day 52 post initial vaccination, micewere intranasally infected with an adenovirus expressing hACE2(Ad/hACE2) to render them permissive. Mice were intranasally inoculatedwith 1.5×10⁵ pfu of SARS-CoV-2 in 50 μL divided between nares.Challenged mice were weighed on the day of infection and daily for up to7 days post infection. At 4- and 7-days post infection, 5 mice weresacrificed from each vaccination and control group, and lungs wereharvested and prepared for pulmonary histology.

The viral titer was quantified by a plaque assay. Briefly, the harvestedlungs were homogenized in PBS using 1.0 mm glass beads (Sigma Aldrich)and a Beadruptor (Omini International Inc.). Homogenates were added toVero E6 near confluent cultures and SARS-CoV-2 virus titers determinedby counting plaque forming units (pfu) using a 6-point dilution curve

At 4 days post infection, placebo-treated mice had 10⁴ SARS-CoV-2pfu/lung, while the mice immunized with BV2363 without MATRIX-M™ had 10³pfu/lung (FIG. 16). The BV2373 with MATRIX-M™ prime-only groups of miceexhibited a dose dependent reduction in virus titer, with recipients ofthe 10 μg BV2373 dose having no detectable virus at day 4 postinfection. Mice receiving 1 μg, 0.1 μg and 0.01 μg BV2373 doses allshowed a marked reduction in titer compared to placebo-vaccinated mice.In the prime/boost groups, mice immunized with 10 μg, 1 μg and 0.1 μgdoses had almost undetectable lung virus loads, while the 0.01 μg groupdisplayed a reduction of 1 log reduction relative to placebo animals.

Weight loss paralleled the viral load findings. Animals receiving asingle dose of BV2373 (0.1 μg, 1 μg, and 10 μg) and MATRIX-M™ showedmarked protection from weight loss compared to the unvaccinated placeboanimals (FIG. 17A). The mice receiving a prime and boost dose withadjuvant also demonstrated significant protection against weight loss atall dose levels (FIGS. 17B-C). The effect of the presence of adjuvant onprotection against weight loss was evaluated. Mice receiving theprime/boost (two doses) plus adjuvant were significantly protected fromweight loss relative to placebo, while the group immunized withoutadjuvant was not (FIG. 17C). These results showed that BV2373 confersprotection against SARS-CoV-2 and that low doses of the vaccineassociated with lower serologic responses do not exacerbate weight lossor demonstrate exaggerated illness.

Lung histopathology was evaluated on days 4 and day 7 post infection(FIG. 18A and FIG. 18B). At day 4 post infection, placebo-immunized miceshowed denudation of epithelial cells in the large airways withthickening of the alveolar septa surrounded by a mixed inflammatory cellpopulation. Periarteriolar cuffing was observed throughout the lungswith inflammatory cells consisting primarily of neutrophils andmacrophages. By day 7 post infection, the placebo-treated mice displayedperibronchiolar inflammation with increased periarteriolar cuffing. Thethickened alveolar septa remained with increased diffuse interstitialinflammation throughout the alveolar septa (FIG. 18B).

The BV2373 immunized mice showed significant reduction in lung pathologyat both day 4 and day 7 post infection in a dose-dependent manner. Theprime only group displays reduced inflammation at the 10 μg and 1 μgdose with a reduction in inflammation surrounding the bronchi andarterioles compared to placebo mice. In the lower doses of theprime-only groups, lung inflammation resembles that of the placebogroups, correlating with weight loss and lung virus titer. Theprime/boost immunized groups displayed a significant reduction in lunginflammation for all doses tested, which again correlated with lungviral titer and weight loss data. The epithelial cells in the large andsmall bronchi at day 4 and 7 were substantially preserved with minimalbronchiolar sloughing and signs of viral infection. The arterioles ofanimals immunized with 10 μg, 1 μg and 0.1 μg doses have minimalinflammation with only moderate cuffing seen with the 0.01 μg dose,similar to placebo. Alveolar inflammation was reduced in animals thatreceived the higher doses with only the lower 0.01 μg dose associatedwith inflammation (FIGS. 18A-18B). These data demonstrate that BV2373reduces lung inflammation after challenge and that even doses andregimens of BV2373 that elicit minimal or no detectable neutralizingactivity are not associated with exacerbation of the inflammatoryresponse to the virus. Furthermore, the vaccine does not cause vaccineassociated enhanced respiratory disease (VAERD) in challenged mice.

T Cell Response

The effect of the vaccine composition comprising a CoV S polypeptide ofSEQ ID NO: 87 on the T cell response was evaluated. BALB/c mice (N=6 pergroup) were immunized intramuscularly with 10 μg BV2373 with or without5 μg MATRIX-M™ in 2 doses spaced 21-days apart. Spleens were collected7-days after the second immunization (study day 28). A non-vaccinatedgroup (N=3) served as a control.

Antigen-specific T cell responses were measured by ELISPOT™ enzymelinked immunosorbent assay and intracellular cytokine staining (ICCS)from spleens collected 7-days after the second immunization (study day28). The number of IFN-γ secreting cells after ex vivo stimulationincreased 20-fold (p=0.002) in spleens of mice immunized with BV2373 andMATRIX-M™ compared to BV2373 alone as measured by the ELISPOT™ assay(FIG. 19). In order to examine CD4+ and CD8+ T cell responsesseparately, ICCS assays were performed in combination with surfacemarker staining. Data shown are gated on CD44hi CD62L-effector memory Tcell population. The frequency of IFN-γ+, TNF-α+, and IL-2+cytokine-secreting CD4+ and CD8+ T cells was significantly higher(p<0.0001) in spleens from mice immunized with BV2373 as compared tomice immunized without adjuvant (FIG. 20A-C and FIG. 21A-C). Further,the frequency of multifunctional CD4+ and CD8+ T cells, whichsimultaneously produce at least two or three cytokines was alsosignificantly increased (p<0.0001) in spleens from the BV2373/MATRIX-M™immunized mice as compared to mice immunized in the absence of adjuvant(FIGS. 20D-E and FIGS. 21D-E). Immunization with BV2373/MATRIX-M™resulted in higher proportions of multifunctional phenotypes (e.g., Tcells that secrete more than one of IFN-γ, TNF-α, and IL-2) within bothCD4+ and CD8+ T cell populations. The proportions of multifunctionalphenotypes detected in memory CD4+ T cells were higher than those inCD8+ T cells (FIG. 22).

Type 2 cytokine IL-4 and IL-5 secretion from CD4+ T cells was alsodetermined by ICCS and ELISPOT™ respectively. Immunization withBV2373/MATRIX-M™ also increased type 2 cytokine IL-4 and IL-5 secretion(2-fold) compared to immunization with BV2373 alone, but to a lesserdegree than enhancement of type 1 cytokine production (e.g. IFN-γincreased 20-fold) (FIGS. 23A-C). These results indicate thatadministration of the MATRIX-M™ adjuvant skewed the CD4+ T celldevelopment toward Th1 responses.

The effect of immunization on germinal center formation was assessed bymeasuring the frequency of CD4+T follicular helper (TFH) cells andgerminal center (GC) B cells in spleens. MATRIX-M™ administrationsignificantly increased the frequency of TFH cells (CD4+CXCR5+PD-1+) wassignificantly increased (p=0.01), as well as the frequency of GC B cells(CD19+GL7+CD95+) (p=0.0002) in spleens (FIGS. 24A-B and FIGS. 25A-B).

Example 3 Immunogenicity of Coronavirus Spike (S) PolypeptideNanoparticle Vaccines in Olive Baboons

The immunogenicity of a vaccine composition comprising BV2373 in baboonswas assessed. Adult olive baboons were immunized with a dose range (1μg, 5 μg and 25 μg) of BV2373 and 50 μg MATRIX-M™ adjuvant administeredby intramuscular (IM) injection in two doses spaced 21-days apart. Toassess the adjuvanting activity of MATRIX-M™ in non-human primates,another group of animals was immunized with 25 μg of BV2373 withoutMATRIX-M™. Anti-S protein IgG titers were detected within 21-days of asingle priming immunization in animals immunized with BV2373/MATRIX-M™across all the dose levels (GMT=1249-19,000). Anti-S protein IgG titersincreased over a log (GMT=33,000-174,000) within 1 to 2 weeks followinga booster immunization (days 28 and 35) across all of the dose levels.(FIG. 26A).

Low levels of hACE2 receptor blocking antibodies were detected inanimals following a single immunization with BV2373 (5 μg or 25 μg) andMATRIX-M™ (GMT=22-37). Receptor blocking antibody titers weresignificantly increased within one to two weeks of the boosterimmunization across all groups immunized with BV2373/MATRIX-M™(GMT=150-600) (FIG. 26B). Virus neutralizing antibodies were elevated(GMT=190-446) across all dose groups after a single immunization withBV2373/MATRIX-M™. Animals immunized with 25 μg BV2373 alone had nodetectable antibodies that block S-protein binding to hACE2 (FIG. 26C).Neutralizing titers were increased 6- to 8-fold one week following thebooster immunization (GMT=1160-3846). Neutralizing titers increased anadditional 25- to 38-fold following the second immunization(GMT=6400-17,000) (FIG. 26C). There was a significant correlation(p<0.0001) between anti-S IgG levels and neutralizing antibody titers(FIG. 27). The immunogenicity of the adjuvanted vaccine in nonhumanprimates is consistent with the results of Example 2 and furthersupports the role of MATRIX-M™ in promoting the generation ofneutralizing antibodies and dose sparing.

PBMCs were collected 7 days after the second immunization (day 28), andthe T cell response was measured by ELISPOT assay. PBMCs from animalsimmunized with BV2373 (5 μg or 25 μg) and MATRIX-M™ had the highestnumber of IFN-γ secreting cells, which was 5-fold greater compared toanimals immunized with 25 μg BV2373 alone or BV2373 (1 μg) and MATRIX-M™(FIG. 28). By ICCS analysis, immunization with BV2373 (5 μg) andMATRIX-M™showed the highest frequency of IFN-γ+, IL-2+, and TNF-α+CD4+ Tcells (FIGS. 29A-C). This trend was also true for multifunctional CD4+ Tcells, in which at least two or three type 1 cytokines were producedsimultaneously (FIGS. 29D-E).

Example 4 Structural Characterization of Coronavirus Spike (S)Polypeptide Nanoparticle Vaccines

Transmission electron microscopy (TEM) and two dimensional (2D) classaveraging were used to determine the ultrastructure of BV2373. Highmagnification (67,000× and 100,000×) TEM images of negatively stainedBV2373 showed particles corresponding to S-protein homotrimers.

An automated picking protocol was used to construct 2D class averageimages (Lander G. C. et al. J Struct Biol. 166, 95-102 (2009); SorzanoC. O. et al., J Struct Biol. 148, 194-204 (2004).). Two rounds of 2Dclass averaging of homotrimeric structures revealed a triangularparticle appearance with a 15 nm length and 13 nm width (FIG. 10, topleft). Overlaying the recently solved cryoEM structure of the SARS-CoV-2spike protein (EMD ID: 21374) over the 2D BV2373 image showed a good fitwith the crown-shaped 51 (NTD and RBD) and the S2 stem (FIG. 10, bottomleft). Also apparent in the 2D images was a faint projection thatprotruded from the tip of the trimeric structure opposite of the NTD/RBDcrown (FIG. 10, top right). 2D class averaging using a larger box sizeshowed these faint projections form a connection between the S-trimerand an amorphous structure. (FIG. 10, bottom right).

Dynamic light scattering (DLS) show that the wild-type CoV S protein hada Z-avg particle diameter of 69.53 nm compared to a 2-fold smallerparticle size of BV2365 (33.4 nm) and BV2373 (27.2 nm). Thepolydispersity index (PDI) indicated that BV2365 and BV2373 particleswere generally uniform in size, shape, and mass (PDI=0.25-0.29) comparedto the wild-type spike-protein (PDI=0.46) (Table 3).

TABLE 3 Particle Size and Thermostability of SARS-CoV-2 Trimeric SpikeProteins Differential Scanning Calorimetry (DSC) Dynamic LightScattering (DLS) SARS-CoV-2 S ΔHcal Z- avg protein T_(max) (° C.)¹(kJ/mol) diameter² (nm) PDI³ Wild-type 58.6 153 69.53 0.46 BV2365 61.3466 33.40 0.25 BV2373 60.4 732 27.21 0.29 ¹T_(max): melting temperature²Z-avg: Z-average particle size ³PDI: polydispersity index

The thermal stability of the S-trimers was determined by differentialscanning calorimetry (DSC). The thermal transition temperature of thewild-type CoV S-protein (T_(max)=58.6° C.) was similar to BV2365 andBV2373 with a T_(max)=61.3° C. and 60.4° C., respectively (Table 3). Ofgreater significance, was the 3-5 fold increased enthalpy of transitionrequired to unfold the BV2365 and BV2373 variants (ΔHcal=466 and 732kJ/mol, respectively) compared to the lower enthalpy required to unfoldthe WT spike protein (ΔHcal=153 kJ/mol). These results are consistentwith improved thermal stability of the BV2365 and BV2373 compared tothat of WT spike protein (Table 3).

The stability of the CoV Spike (S) polypeptide nanoparticle vaccines wasevaluated by dynamic light scattering. Various pHs, temperatures, saltconcentrations, and proteases were used to compare the stability of theCoV Spike (S) polypeptide nanoparticle vaccines to nanoparticle vaccinescontaining the native CoV Spike (S) polypeptide.

Example 5 Stability of Coronavirus Spike (S) Polypeptide NanoparticleVaccines

The stability of the CoV Spike (S) polypeptide nanoparticle vaccines wasevaluated by dynamic light scattering. Various pHs, temperatures, saltconcentrations, and proteases were used to compare the stability of theCoV Spike (S) polypeptide nanoparticle vaccines to nanoparticle vaccinescontaining the native CoV Spike (S) polypeptide. The stability of BV2365without the 2-proline substitutions and BV2373 with two prolinessubstitution was assessed under different environmental stressconditions using the hACE2 capture ELISA. Incubation of BV2373 at pHextremes (48 hours at pH 4 and pH 9), with prolonged agitation (48hours), and through freeze/thaw (2 cycles), and elevated temperature (48hours at 25° C. and 37° C.) had no effect on hACE2 receptor binding(IC50=14.0-18.3 ng mL-1).

Oxidizing conditions with hydrogen peroxide reduced binding of hACE2binding to BV2373 8-fold (IC50=120 ng mL-1) (FIG. 12A). BV2365 withoutthe 2-proline substitutions was less stable as determined by asignificant loss of hACE2 binding under multiple conditions (FIG. 12B).

The stability of BV2384 (SEQ ID NO: 110) and BV2373 (SEQ ID NO: 87) werecompared. BV2384 has a furin cleavage site sequence of GSAS (SEQ ID NO:97), whereas BV2373 has a furin cleavage site of QQAQ (SEQ ID NO: 7). Asdemonstrated by SDS-PAGE and Western Blot, BV2384 showed extensivedegradation in comparison to BV2373 (FIG. 32). Furthermore, scanningdensitometry and recovery data demonstrate the unexpected loss of fulllength CoV S protein BV2384, lower purity, and recovery (FIG. 33) incomparison to BV2373 (FIG. 34).

Example 6 Immune Response in Cynomolgus Macaques

We assessed the immune response induced by BV2373 in a Cynomolgusmacaque model of SARS-CoV-2 infection. Groups 1-6 were treated as shownin Table 4.

TABLE 4 Groups 1-6 of Cynomolgus macaque study MATRIX- Immuni- BloodGroup BV2373 M^(Tm) zation Draw Challenge (N = 4) Dose Dose (Days)(days) (Day) 1 Placebo — 0, 21 0, 21, 33 35 2 2.5 μg 25 μg 0, 21 0, 21,33 35 3   5 μg 25 μg 0 0, 21, 33 35 4   5 μg 50 μg 0, 21 0, 21, 33 35 5  5 μg 50 μg 0 0, 21, 33 35 6  25 μg 50 μg 0, 21 0, 21, 33 35

Administration of a vaccine comprising BV2373 resulted in the inductionof anti-CoV-S antibodies (FIG. 35A) including neutralizing antibodies(FIG. 35B). Anti-CoV-S antibodies were induced after administration ofone (FIG. 38A) or two doses (FIG. 38B) of BV2373. Administration of thevaccine comprising BV2373 also resulted in the production of antibodiesthat blocked binding of the CoV S protein to hACE2 (FIG. 38C and FIG.38D). There was a significant correlation between anti-CoV S polypeptideIgG titer and hACE2 inhibition titer in Cynomolgus macaques afteradministration of BV2373 (FIG. 38E). The ability of BV2373 to induce theproduction of neutralizing antibodies was evaluated by cytopathic effect(CPE) (FIG. 40A) and plaque reduction neutralization test (PRNT) (FIG.40B). The data revealed that vaccine formulations of Table 4 producedSARS-CoV-2 neutralizing titers, in contrast to the control.

The vaccine comprising BV2373's ability to induce anti-CoV-S antibodiesand antibodies that block binding of hACE2 to the CoV S protein inCynomolgus macaques was compared to human convalescent serum. The datarevealed that the BV2373 vaccine formulation induced superior anti-CoV Spolypeptide and hACE2 inhibition titers as compared to humanconvalescent serum (FIG. 39).

The BV2373 vaccine formulation also caused a decrease of SARS-CoV-2viral replication (FIGS. 36A-B). Viral RNA (FIG. 36A, corresponding tototal RNA present) and viral sub-genomic RNA (sgRNA) (FIG. 36B,corresponding to replicating virus) levels were assessed in bronchiolarlavage (BAL) at 2 days and 4 days post-challenge with infectious virus(d2pi and d4pi). Most subjects showed no viral RNA. At Day 2 smallamounts of RNA were measured in some subjects. By Day 4, no RNA wasmeasured except for two subjects at the lowest dose of 2.5 μg.Sub-genomic RNA was not detected at either 2 days or 4 days except for 1subject, again at the lowest dose. Viral RNA (FIG. 37A) and viralsub-genomic (sg) RNA (FIG. 37B) were assessed by nasal swab at 2 daysand 4 days post-infection (d2pi and d4pi). Most subjects showed no viralRNA. At Day 2 and Day 4 small amounts of RNA were measured in somesubjects. Sub-genomic RNA was not detected at either 2 Days or 4 days.Subjects were immunized Day 0 and in the groups with two doses Day 0 andDay 21. These data show that the vaccine decreases nose total virus RNAby 100-1000 fold and sgRNA to undetectable levels, and confirm thatimmune response to the vaccine will block viral replication and preventviral spread.

Example 7 Evaluation of CoV S Polypeptide Nanoparticle Vaccines inHumans

We assessed the safety and efficacy of a vaccine comprising BV2373 in arandomized, observer-blinded, placebo-controlled Phase 1 clinical trialin 131 healthy participants 18-59 years of age. Participants wereimmunized with two intramuscular injections, 21 days apart. Participantsreceived BV2373 with or without MATRIX-M™ (n=106) or placebo (n=25).Groups A-E were treated as shown in Table 5. FIG. 41 shows a timeline ofthe evaluation of clinical endpoints.

TABLE 5 Groups A-E of Phase 1 Human Study Group Participants Day 0 Day21 (+5 days) (N = Random- BV2373 MATRIX- BV2373 MATRIX- 25) izedSentinel Dose M ™ Dose Dose M ™ Dose A 25 —  0 μg  0 μg 0 μg 0 μg B 25 —25 μg  0 μg 25 μg  0 μg C 25 3  5 μg 50 μg 5 μg 50 μg  D 25 3 25 μg 50μg 25 μg  50 μg  E 25 — 25 μg 50 μg 0 μg 0 μg

Overall reactogenicity was mild, and the vaccinations were welltolerated. Local reactogenicity was more frequent in patients treatedwith BV2373 and MATRIX-M™ (FIGS. 42A-B).

The immunogenicity of BV2373 with and without MATRIX-M™ was evaluated.21 days after vaccination, anti-CoV-S antibodies were detected for allvaccine regimens (FIG. 43A). Geometric mean fold rises (GMFR) in vaccineregimens comprising MATRIX-M™ exceeded those induced by unadjuvantedBV2373. 7 days after a second vaccination (day 28), the anti-CoV-S titerincreased an additional eight-fold over responses seen with firstvaccination and within 14 days (Day 35) responses had more than doubledyet again, achieving GMFRs approximately 100-fold over those observedwith BV2373 alone. A single vaccination with BV2373/MATRIX-M™ achievedsimilar anti-CoV-S titer levels to those in asymptomatic (exposed)COVID-19 patients. A second vaccination achieved GMEU levels thatexceeded convalescent serum from outpatient-treated COVID-19 patients bysix-fold, achieved levels similar to convalescent serum from patientshospitalized with COVID-19, and exceeded overall convalescent serumanti-CoV-S antibodies by nearly six-fold. The responses in the two-dose5-μg and 25-μg BV2373/MATRIX-M™ regimens were similar. This highlightsthe ability of the adjuvant (MATRIX-M™) to enable dose sparing.

Neutralizing antibodies were induced in all groups treated with BV2373(FIG. 43B). Groups treated with BV2373 and MATRIX-M™ regimens exhibitedan approximately five-fold GMFR than groups treated with BV2373 alone(FIG. 43B). Second vaccinations with adjuvant had a profound effect onneutralizing antibody titers—inducing>100 fold rise over singlevaccinations without adjuvant. When compared to convalescent serum,second vaccinations with BV2373/MATRIX-M™ achieved GMT levels four-foldgreater than outpatient-treated COVID-19 patients, levels spanning thoseof patients hospitalized with COVID-19, and exceeded overallconvalescent serum GMT by four fold.

Convalescent serum, obtained from COVID-19 patients with clinicalsymptoms requiring medical care, demonstrated proportional anti-CoV-SIgG and neutralization titers that increased with illness severity(FIGS. 43A-B).

A strong correlation was observed between neutralizing antibody titersand anti-CoV-S IgG in patients treated with BV2373 and MATRIX-M™(r=0.9466, FIG. 44C) similar to that observed in patients treated withconvalescent sera (r=0.958) (FIG. 44A). This correlation was notobserved in subjected administered unadjuvanted BV2373 (r=0.7616) (FIG.44B). Both 5 μg and 25 μg BV2373/MATRIX-M™ groups (groups C-E of Table5) demonstrated similar magnitudes of two-dose responses and everyparticipant seroconverted using either assay measurement when a two-doseregimen was utilized.

T-cell responses in 16 participants (four participants from each ofGroups A through D) showed that BV2373/MATRIX-M™ regimens inducedantigen-specific polyfunctional CD4+ T-cell responses in terms of IFN-γ,IL-2, and TNF-α production upon stimulation with BV2373. There was astrong bias toward production of Th1 cytokines (FIGS. 45A-D).

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes. However, mention of any reference,article, publication, patent, patent publication, and patent applicationcited herein is not, and should not be taken as, an acknowledgment orany form of suggestion that they constitute valid prior art or form partof the common general knowledge in any country in the world.

1. An immunogenic composition comprising: (a) a non-naturally occurringsudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (CoVS) glycoprotein, wherein the CoV S glycoprotein consists of twomodifications compared to a native CoV S glycoprotein wherein (i) thefirst modification is an inactivated furin cleavage site consisting ofmutation of amino acids 669-672 to QQAQ (SEQ ID NO: 7); and (ii) thesecond modification is mutation of amino acids 973 and 974 to proline;wherein the amino acids are numbered according to SEQ ID NO: 2; (b) asaponin adjuvant; and (c) a pharmaceutically acceptable buffer.
 2. Theimmunogenic composition of claim 1, comprising between about 5 μg andabout 25 μg of CoV S glycoprotein.
 3. The immunogenic composition ofclaim 2, comprising about 5 μg of CoV S glycoprotein.
 4. The immunogeniccomposition of claim 1, comprising about 3 μg of CoV S glycoprotein. 5.The immunogenic composition of claim 1, comprising about 50 μg ofsaponin adjuvant.
 6. The immunogenic composition of claim 1, wherein theCoV S glycoprotein forms a nanoparticle comprising the CoV Sglycoprotein and a non-ionic detergent core.
 7. The immunogeniccomposition of claim 6, wherein the non-ionic detergent core is selectedfrom the group consisting of polysorbate-20 (PS20), polysorbate-40(PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80(PS80).
 8. The immunogenic composition of claim 7, wherein the non-ionicdetergent core is PS80.
 9. The immunogenic composition of claim 1,wherein the saponin adjuvant comprises: a first iscom particlecomprising fraction A of Quillaja Saponaria Molina and not fraction C ofQuillaja Saponaria Molina; and (ii) a second iscom particle comprisingfraction C of Quillaja Saponaria Molina and not fraction A of QuillajaSaponaria Molina.
 10. The immunogenic composition of claim 9, whereinfraction A of Quillaja Saponaria Molina and fraction C of QuillajaSaponaria Molina account for about 85% by weight and about 15% byweight, respectively, of the sum of the weights of fraction A ofQuillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina inthe saponin adjuvant.
 11. A method of stimulating an immune responseagainst SARS-CoV-2 in a subject comprising administering the immunogeniccomposition of claim
 1. 12. The method of claim 11, comprising betweenabout 5 μg and about 25 μg of CoV S glycoprotein.
 13. The method ofclaim 12, comprising about 5 μg of CoV S glycoprotein.
 14. The method ofclaim 11, comprising about 3 μg of CoV S glycoprotein.
 15. The method ofclaim 11, comprising about 50 μg of saponin adjuvant.
 16. The method ofclaim 11, wherein the saponin adjuvant comprises: (i) a first iscomparticle comprising fraction A of Quillaja Saponaria Molina and notfraction C of Quillaja Saponaria Molina; and (ii) a second iscomparticle comprising fraction C of Quillaja Saponaria Molina and notfraction A of Quillaja Saponaria Molina.
 17. The method of claim 16,wherein fraction A of Quillaja Saponaria Molina and fraction C ofQuillaja Saponaria Molina account for about 85% by weight and about 15%by weight, respectively, of the sum of the weights of fraction A ofQuillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina inthe saponin adjuvant.
 18. The method of claim 11, wherein the CoV Sglycoprotein forms a nanoparticle comprising the CoV S glycoprotein anda non-ionic detergent core.
 19. The method of claim 18, wherein thenon-ionic detergent core is selected from the group consisting ofpolysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60),polysorbate-65 (PS65), and polysorbate-80 (PS80).
 20. The method ofclaim 19, wherein the non-ionic detergent core is PS80.
 21. The methodof claim 11, wherein the subject is administered a first dose at day 0and a boost dose at day
 21. 22. The method of claim 11, wherein a singledose of the immunogenic composition is administered.
 23. The method ofclaim 11, comprising administering a second immunogenic compositionwithin about 30 days before or after the first immunogenic compositionis administered.
 24. The method of claim 23, wherein the first andsecond immunogenic compositions are the same.
 25. The method of claim23, wherein the first and second immunogenic compositions are different.26. The method of claim 23, wherein the first immunogenic composition isthe immunogenic composition of claim 1 and the second immunogeniccomposition is selected from the group consisting of an mRNA encoding aSARS-CoV-2 Spike glycoprotein, a plasmid DNA encoding a SARS-CoV-2 Spikeglycoprotein, a viral vector encoding a SARS-CoV-2 Spike glycoprotein,and an inactivated SARS-CoV-2 virus.
 27. The method of claim 23, whereinthe first immunogenic composition is selected from the group consistingof an mRNA encoding a SARS-CoV-2 Spike glycoprotein, a plasmid DNAencoding a SARS-CoV-2 Spike glycoprotein, a viral vector encoding aSARS-CoV-2 Spike glycoprotein, and an inactivated SARS-CoV-2 virus andthe second immunogenic composition is the immunogenic composition ofclaim 1.